Method for the diagnosis and prognosis of malignant diseases

ABSTRACT

Methods for the treatment of tumors and cancer by exploiting the surface expression of the usually nuclear-localized protein, nucleolin.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/118,854, filed Apr. 8, 2002, which is herein incorporated byreference in its entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This subject matter of this application may have been funded in partunder the following research grants and contracts: National Institutesof Health 1 R21 CA 91115, and Department of Defense DAMD17-98-1-8583 andDAMD17-01-1-0067. The U.S. Government may have rights in this invention.

BACKGROUND

Cancer

Being well-prepared for battle engenders success; when the foe iscancer, early detection results in a greater likelihood that medicalintervention will be successful. At early stages, treatments can oftenbe targeted only to the affected tissues, diminishing side effects. Ifnot caught early, cancer cells may metastasize and spread throughout thebody. The prognosis in this case is more dire, and medical treatmentsare often applied systemically, killing not only cancer cells, but largenumbers of healthy cells.

The National Cancer Institute estimates that in 2002, 1.285 millionAmericans will be newly diagnosed with cancer, and more than 560,000Americans will die from of cancer related illness.

A hallmark of a cancer cell is uncontrolled proliferation. Uncontrolledproliferation of these cells can manifest as cell masses (tumors) thatinterfere with normal organ function. If proliferation is not controlledor contained, cells from tumors migrate and colonize other tissues ofthe body, eventually resulting in death.

External factors, such as tobacco smoke, radiation and viruses, can leadto alterations in specific genes that result in unregulated cellularproliferation. Intrinsic factors, including inheritable mutations,hormone levels and metabolism, contribute to one's risk of contractingcancer.

Cancer cells also exhibit morphological and functional aberrations.Cellular morphology may be less organized; for example, the cells losingthe asymmetric organelle and structural organization (cell polarity)that allows for proper cell function. Cell-cell and cell-substratumcontacts, the specificities of which are also necessary for normalfunction, are often modulated or lost. Functionally, the cells may carryon few, if any, wild-type functions, or may have exaggerated,unregulated normal functions, such as hormone secretion. Such cellsregress to early developmental stages, appearing less differentiatedthan their wild-type (i.e., normal) parents.

Cancer cells also often mis-express or mis-target proteins toinappropriate cellular compartments. Proteins may be up- ordown-regulated; even proteins not usually expressed by a specific celltype can be expressed by the transformed counterpart. Proteinmis-expression can have a plethora of downstream cellular effects,including drastic changes in membrane composition, organelle formation,or physiology. Mis-targeting of proteins (and other molecules, such aslipids, etc.) also contributes to the loss of cell polarity.

Treatments for Cancer

Methods for treating cancers include surgery (physical removal of thecancerous tissues), radiation therapy (killing cells by exposure tocell-lethal doses of radioactivity), chemotherapy (administeringchemical toxins to the cells), immunotherapy (using antibodies thattarget cancer cells and mark them for destruction by the innate immunesystem) and nucleic acid-based therapies (e.g., expression of geneticmaterial to inhibit cancer growth). Each approach, however, has itslimitations.

Surgery, chemotherapy and radiation therapy suffer from similarsignificant limitations, such as incomplete removal of cancer cells orthe inadvertent killing of healthy cells. Surgical tactics are mosteffective when the cancers are in early stages and limited localizedarea in the body. Even in the few cases that are diagnosed early,surgical removal of cancerous cells is often incomplete, andre-emergence of metastatic lesions often follow. When chemotherapeuticagents are administered in precise doses, they are preferentially toxicto rapidly proliferating cancer cells and not injuring the majority ofhealthy cells. Locally delivered doses of external beam radiation aremost effective on rapidly growing cells, killing them by introducingnon-specific, DNA-damage. Radiation therapy, like surgery, works bestwhen the targeted cancer mass is well delimited; the balance of killinghealthy cells versus cancer cells must be carefully weighed. Bothchemotherapy and radiation therapy are not entirely selective forcancerous cells; inevitably, some healthy cells fall victim to the toxiceffects, inflicting profound side-effects on the already-sufferingcancer victim.

Other common approaches, immunotherapy and gene therapy, can be quitepowerful and surpass surgery, chemo- and radiation therapy. Thesetechniques target specific factors that are associated with tumorsurvival, cell growth or metastasis. For example, antibodies can targetspecific tumor-associated proteins, such as the monoclonal antibody thatbinds to a surface protein specific to the B-cells, CD20 (RITUXAN®;Genentech, Inc. and IDEC Inc.) that is used to treat B-cellmalignancies. An example of an effective gene therapy is anti-senseinhibition of bcl-2 expression (GENASENSE®; Genta, Inc.). Whileeffective, the challenge is to identify those clinically relevant genesand proteins and develop appropriate therapeutics that target them toresult in the destruction of the cancer cell. Furthermore, the processis not only laborious in identifying these molecules, but in manyinstances, the identified molecules will be specific to only one type ofcancer or tumor cell.

SUMMARY

The invention provides methods for treating tumors or cancer in asubject by:

(1) administering a therapeutically effective amount of ananti-nucleolin agent and a pharmaceutically acceptable carrier. Thispharmaceutical composition may further comprise other chemotherapeuticor chemotoxic agents, such as cyclophosphamide, etoposide, doxorubicin,methotrexate, vincristine, procabazine, prednizone, dexamethasone,tamoxifen citrate, carboplatin, cisplatin, oxaliplatin, 5-fluorouracil,camptothecin, zoledronic acid, Ibandronate and mytomycin. Inconjunction, radiation therapy may also be plied.

(2) administering a therapeutically effective amount of ananti-nucleolin antibody and a pharmaceutically acceptable carrier,wherein the antibody is substantially non-immunogenic to human. Thispharmaceutical composition may further comprise other chemotherapeuticor chemotoxic agents. In conjunction, radiation therapy may also beplied.

(3) administering a therapeutically effective amount of ananti-nucleolin antibody, a chemotoxic or chemotherapeutic agent and apharmaceutically acceptable carrier.

(4) administering a therapeutically effective amount of a nucleolinantibody and a pharmaceutically acceptable carrier, and further treatingthe subject with radiation therapy.

(5) administering a therapeutically effective amount of a duplexinterfering RNA to nucleolin and a pharmaceutically acceptable carrier.

(6) administering a nucleolin anti-sense molecule which inhibits theproduction of the nucleolin protein.

(7) administering a nucleolin-interfering RNA molecule which inhibitsthe expression of the nucleolin gene.

In these aspects, a chemotherapeutic or chemotoxic agent may becyclophosphamide, etoposide, doxorubicin, methotrexate, vincristine,procabazine, prednizone, dexamethasone, tamoxifen citrate, carboplatin,cisplatin, oxaliplatin, 5-fluorouracil, camptothecin, zoledronic acid,Ibandronate and mytomycin.

In yet another aspect, the invention provides pharmaceuticalcompositions that comprise a pharmaceutically acceptable carrier and:

(8) an anti-sense oligonucleotide directed against nucleolin.

(9) an inhibitory RNA against nucleolin.

(10) a nucleolin antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows nuclear nucleolin staining in various cell lines. Shown areimmunofluorescent (B, D, F, H) and parallel phase contrast micrographs(A, C, E, G). Cell lines that were analyzed were: DU145 prostate cancercells (A, B), MDA-MB-231 breast cancer cells (C, D), HeLa cervicalcancer cells (E, F) and HS27 normal skin cells (G, H). An anti-nucleolinantibody was used; the cells were permeabilized before staining to allowthe antibody access to the cytoplasmic and nuclear compartments.

FIG. 2 shows plasma membrane nucleolin staining in the cell lines shownin FIG. 1. Shown are immunofluorescent (B, D, F, H) and parallel phasecontrast micrographs (A, C, E, G). Cell lines that were analyzed were:DU145 prostate cancer cells (A, B), MDA-MB-231 breast cancer cells (C,D), HeLa cervical cancer cells (E, F) and HS27 normal skin cells (G, H).An anti-nucleolin antibody was used; the cells were not permeabilizedbefore staining, allowing the antibody access to only the plasmamembrane.

FIG. 3 shows the comparative proliferation rates of cell lines asmeasured by MTT assay. Square, DU145; diamonds, HeLa; circles, HS27.Although MDA-MB-231 was not included in this experiment, proliferationrates for these four cell lines have been determined to beDU145>MDA-MB-231>HeLa>HS27. Note that the cell lines with high levels ofplasma membrane nucleolin correspond to those with the most rapidproliferation (DU145 and HeLa; see FIG. 2).

FIG. 4 shows phase contrast (B,D) and immunofluorescent image (A,C) of aparaffin-embedded specimen resected from a patient with squamous cellcarcinoma of the head and neck. The specimen was stained for plasmamembrane nucleolin and counter-stained with propidium iodide to showcell nuclei. Images (C) and (D) show images (A) and (B) overlaid withmarkings to better show nucleolin staining. The area 1 encompassed bythe white line includes intense nucleolin staining, while the areasoutside of 1 show little 3 or no 2 signal.

FIG. 5 shows phase contrast (B, D) and immunofluorescent images (A, C)of small cell (NCI-H82) and non-small cell lung (NCI-HI299) cancer celllines placed onto a microscope slide using a cytospinner. Samples werestained for plasma membrane nucleolin and counter-stained with propidiumiodide to show cell nuclei. Cells with exceptionally well-stained plasmamembranes are denoted by asterisks (*).

FIG. 6 shows phase contrast (B, D, F) and immunofluorescent images (A,C, E) of peripheral blood (A, B) or bone marrow (C, D and E, F) fromhuman subjects. Samples were stained for plasma membrane nucleolin andcounter-stained with propidium iodide to show cell nuclei. Highlystained cells for nucleolin are marked with an asterisk (*); these wereonly seen in those patients suffering from carcinomas (A,B and C,D),while cells from a healthy patient did not display any plasma membranenucleolin staining (E, F).

DETAILED DESCRIPTION

The invention is based on the discovery of a correlation betweennucleolin plasma membrane expression and the presence and aggressivenessof neoplastic cells. The unexpected discovery that nucleolin, mostlyrestricted to the interior of the healthy cell nucleus, when found onthe cell surface, correlates with a pre-malignant or malignantphenotype. Not only does this observation facilitate cancer diagnosisand prognosis, but also provides a novel and powerful treatmentstrategy. The invention provides methods of treating cancer byadministering a compound that specifically targets nucleolin.Furthermore, the invention provides methods for treating cancer byadministering a nucleolin-targeting compound in conjunction with othercancer therapies, e.g., an anti-cancer drug. Such combination therapyachieves superior and synergistic therapeutic results.

The advantages of using surface-localized nucleolin to treat tumors andcancers include:

(1) Specificity. Plasma membrane nucleolin is not usually observed inthe plasma membrane of most wild-type (healthy) cells. Thus, unlikeother non-specific therapeutic approaches (e.g., surgery, radiation,chemotoxins), plasma membrane nucleolin targeting can be used tospecifically kill cancer cells.

(2) Broad applicability. Unlike previous immuno- and gene therapies,nucleolin expression on the plasma membrane occurs on many types ofcancer cells. Many different cancers, therefore, may be treated byexploiting plasma membrane nucleolin; yet, unlike other less-specifictreatments (e.g., radiation therapy), healthy cells are not damaged orkilled.

(3) Treatment: early or late. Because plasma membrane nucleolin isindicative of not only malignant cells, but also of pre-malignant cells,treatment can commence with the detection of surface nucleolin, evenbefore a tumor mass would usually be detected by other means.

While investigating the anti-proliferative activity of non-anti-senseguanosine-rich oligonucleotides (GROs) on cancer cells, it was foundthat such anti-proliferative GROs bind nucleolin to exert their effects(Bates et al., 1999; Miller et al., 2000). Nucleolin (Bandman et al.,1999) is an abundant, non-ribosomal protein of the nucleolus, the siteof ribosomal gene transcription and packaging of pre-ribosomal RNA. This707 amino acid phosphoprotein has a multi-domain structure consisting ofa histone-like N-terminus, a central domain containing four RNArecognition motifs and a glycine/arginine-rich C-terminus and has anapparent molecular weight of 110 kD. Its multiple domain structurereflects the remarkably diverse functions of this multifaceted protein(Ginisty et al., 1999; Srivastava and Pollard, 1999; Tuteja and Tuteja,1998). Nucleolin has been implicated in many fundamental aspects of cellsurvival and proliferation. Most understood is the role of nucleolin inribosome biogenesis. Other functions may include nucleocytoplasmictransport, cytokinesis, nucleogenesis and apoptosis. Nucleolin is one ofthe nuclear organizer region (NOR) proteins whose levels, as measured bysilver staining, are assessed by pathologists as a marker of cellproliferation and an indicator of malignancy (Derenzini, 2000).

Also present in the cell plasma membrane in a few cell types, such aslymphocytes and inner medullary collecting duct cells, nucleolin hasbeen hypothesized to function as a receptor (e.g., (Callebaut et al.,1998; Sorokina and Kleinman, 1999). However, the role of plasma membranenucleolin is not well understood. In addition, it is not clear whetherthe plasma membrane nucleolin is identical to the nucleolar protein, orif it represents a different isoform or nucleolin-like protein. However,the expression of plasma membrane nucleolin is specific to neoplasticcells (such as malignant or pre-malignant); thus the function of plasmamembrane nucleolin need not be known for effective therapeuticintervention.

Definitions

Neoplasm, Malignancy, Tumor, Cancer Cells

A neoplasm is an abnormal tissue growth resulting from neoplastic cells,cells that proliferate more rapidly and uncontrollably than normalcells. Usually partially or completely structurally disorganized,neoplasms lack functional coordination with the corresponding normaltissue. Neoplasms usually form a distinct tissue mass that may be eitherbenign (tumor) or malignant (cancer).

Cancer cells invade surrounding tissues, may metastasize to distantsites, and are likely to recur after attempted removal, causing death ofa subject if not adequately treated. In addition to structuraldisorganization, cancer cells usually regress to more primitive orundifferentiated states (anaplasia), although morphologically andbiochemically, they may still exhibit many functions of thecorresponding wild-type cells. Carcinomas are cancers derived fromepithelia; sarcomas are derived from connective tissues. In some cases,cancers may not be associated with a tumor, but like the affectedtissue, is defuse, e.g., leukemias.

Cancers may be more aggressive or less aggressive. The aggressivephenotype of a cancer cell refers to the proliferation rate and theability to form tumors and metastasize in nude mice. Aggressive cancersproliferate more quickly, more easily form tumors and metastasize thanless-aggressive tumors.

Tumors and cancers include solid, dysproliferative tissue changes anddiffuse tumors. In the sense that cancers and tumors are abnormalgrowths having uncontrolled proliferation of cells that do not serve anormal physiological function, the terms “tumor” and “cancer” are usedinterchangeably. Examples of tumors and cancers include melanoma,lymphoma, plasmocytoma, sarcoma, glioma, thymoma, leukemia, breastcancer, prostate cancer, colon cancer, liver cancer, esophageal cancer,brain cancer, lung cancer, ovary cancer, cervical cancer, hepatoma, andother neoplasms. For more examples of tumors and cancers, see, forexample (Stedman, 2000).

“Stromal cells” are accessory cells found within a tumor. Such cells maybe, for example, fibroblasts, reticular cells and endothelial cells, andplay a supportive role in tumor growth but are healthy cells. Stromalcells and fibroblasts therefore are constituents of themicro-environment in which tumor cells invade during metastasis.

Neoplastic State

The term “neoplastic state” refers to three conditions: normal,pre-malignant and malignant. “Normal” refers to a growth or cell that isclinically normal (healthy). “Pre-malignant” refers to a growth or cellthat is on the pathway to malignancy, but at the time of examination,would not be classified as malignant by conventional methods.“Malignant” refers to a cell or growth that has at least one of thefollowing properties: locally invasive, destructive growth andmetastasis.

GROs and Other Polypeptide-binding Oligonucleotides

Oligonucleotides are available that specifically bind to polypeptides,such as nucleolins. Examples of such are GROs, which are guanosine-richoligonucleotides. Characteristics of GROs include:

(1) having at least 1 GGT motif

(2) preferably having 4–100 nucleotides, although GROs having many morenucleotides are possible

(3) having chemical modifications to improve stability.

Especially useful GROs form G-quartet structures, as indicated by areversible thermal denaturation/renaturation profile at 295 nm (Bates etal., 1999). Preferred GROs also compete with a telomere oligonucleotidefor binding to a target cellular protein in an electrophoretic mobilityshift assay (Bates et al., 1999).

Other oligonucleotides may have high binding specificity for nucleolin.

Anti-nucleolin Agent

An “anti-nucleolin agent” binds to nucleolin. Examples includeanti-nucleolin antibodies and certain oligonucleotides.

Nucleic Acid-based Definitions

A “structural gene” or “gene” refers to a DNA sequence that istranscribed into messenger RNA (mRNA) which can be translated into apolypeptide (a polypeptide consists of at least two amino acidresidues).

A promoter is a DNA sequence that specifies the site of initiation ofRNA transcription, the direction of transcription, and the rate oftranscription. For this reason, promoters are usually located 5′ of thestart site (designated as +1) for the DNA sequence that encodes theresultant RNA transcript. Promoters can be unregulated or regulated.When a promoter is unregulated, it operates constitutively at aparticular basal level of activity. When the promoter is regulated, theefficiency of a promoter can be modulated in response to an agent. RNAtranscription is increased relative to the basal transcription levelunder circumstances where an agent positively regulates promoteractivity; conversely, RNA transcription is decreased relative to thebasal transcription level under circumstances where an agent negativelyregulates promoter activity. Agents that positively regulate promoteractivity are called activators; whereas agents that negatively regulatepromoter activity are called repressors.

An enhancer is a DNA transcription element that can increase theefficiency of promoter activity. Like promoters, enhancers arephysically linked to the affected gene. Enhancers may also beunregulated or regulated. Unlike promoters, however, enhancers cannotspecify the start site of RNA transcription or the direction oftranscription. Enhancers can stimulate gene expression independent ofthe enhancer's orientation and location with respect to the start siteof RNA transcription. Because enhancers do not specify the start site ofRNA transcription, enhancers can exert their effects over greatdistances (several kilobases) with respect to a particular gene.

A regulatory sequence is typically a short DNA motif that positively ornegatively responds to the activity of an agent. A regulatory sequencemay be bidirectional or unidirectional. A regulatory sequence can bepart of the modular organization of either a promoter or an enhancer. Inthe context of a promoter, a regulatory sequence either modulatespromoter efficiency and/or affects the selection of initiation sites ofRNA transcription. In the context of an enhancer, a regulatory sequencemodulates the efficiency of an enhancer.

A “cloning vector” is a DNA molecule such as a plasmid, cosmid, orbacteriophage that has the ability to replicate in a cell. Cloningvectors typically contain restriction endonuclease recognition sitesthat enable the introduction of changes and additions of DNA fragments.Cloning vectors also typically include promoters to enable efficientexpression and selectable markers that confer resistance to compoundssuch as ampicillin or tetracycline.

An “expression vector” is a polynucleotide comprising a coding sequence(such as a gene) that is made to be expressed by the host cell.Expression vectors typically contain promoters, enhancers and tissuespecific regulatory elements that are operably linked to the expressedgene or DNA fragment.

The term “isoform” refers to polypeptides that differ in amino acidsequence or post-translational modifications (such as glycosylation orproteolytic processing events). Isoforms are also used to refer topolypeptides arising from a common gene which result from alternativesplicing.

Therapy-related Definitions

“Cytotoxic agent” refers to a substance that inhibits or prevents atleast one function of a cell, or causes the destruction of a cell.Radioactive isotopes (e.g., ²¹¹At, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²Pand radioactive isotopes of Lu), and chemotherapeutic agents and toxins,such as small molecule toxins, or toxins from bacteria, fungi, plants oranimals are examples of such agents.

“Radiation therapy” and “radiotherapy” refers to the use of locallydelivered doses of external beam radiation to effect killing of a tumoror cancer cell.

A “chemotherapeutic agent” is a chemical compound that can be usedeffectively to treat a cancer cell. Examples of commonly used oncologydrugs and agents include vinorelbine (Navelbine®, mytomycin,camptothecin, cyclyphosphamide (Cytoxin®), methotrexate, tamoxifencitrate, 5-fluorouracil, irinotecan, doxorubicin, flutamide, paclitaxel(Taxol®), docetaxel, vinblastine, imatinib mesylate (Gleevec®),anthracycline, letrozole, arsenic trioxide (Trisenox®), anastrozole,triptorelin pamoate, ozogamicin, irinotecan hydrochloride (Camptosar®),BCG,live (Pacis®), leuprolide acetate implant (Viadur), bexarotene(Targretin®), exemestane (Aromasin®), topotecan hydrochloride(Hycamtin®), gemcitabine HCL(Gemzar®), daunorubicin hydrochloride(Daunorubicin HCL®), gemcitabine HCL (Gemzar®), toremifene citrate(Fareston), carboplatin (Paraplatin®), cisplatin (Platinol® andPlatinol-AQ®) oxaliplatin and any other platinum-containing oncologydrug.

“Medicament,” “therapeutic composition” and “pharmaceutical composition”are used interchangeably to indicate a compound, matter, mixture orpreparation that exerts a therapeutic effect in a subject.

“Approved therapeutic antibodies” include rituximab (Rituxan®),gemtuzumab (Mylotarg®), alemtuzumab (Campath®) and trastuzumab(Herceptin®).

“Antibody” is used in the broadest sense and refers to monoclonalantibodies, polyclonal antibodies, multispecific antibodies, antibodyfragments and derivatives.

An “artificial antibody” is a binding agent having polypeptide bindingdomains connected to polypeptide scaffolds (Irving et al., 2001; Koide,2002).

A “conjugated antibody drug” refers to a therapeutic agent whichincludes an antibody or antibody fragment which is conjugated to amoiety that is a cytotoxic agent.

An “antibody fusion protein” refers to a recombinant molecule thatcomprises one or more antibody components and a therapeutic agent suchas a cytokine, enzyme or a cytotoxic agent.

The term “naked antibody” refers to an entire, intact antibody, such asa monoclonal, recombinant monoclonal or polyclonal antibody that isneither fused nor conjugated to an enzyme, cytotoxic agent orchemotherapeutic agent.

A “nucleolin antibody” or “anti-nucleolin antibody” is an antibody,conjugated antibody drug, antibody fusion protein, naked antibody orartificial antibody that binds to nucleolin polypeptides.

“Anti-sense oligonucleotides,” “oligo,” “oligo nucleic acid,”“anti-sense,” or “anti-sense polynucleotide” are sequence-specific drugscapable of selectively modifying or silencing the expression of genes,causing a desired therapeutic effect.

The term “interfering RNA,” “RNAi,” “short interfering RNA” or “doublestranded interfering RNA” refer to either free ribonucleic acidmolecules or those generated in vivo by means of gene expressionsystems. Such interfering RNA molecules are able to alter the expressionof a target gene.

“Treating a tumor” or “treating a cancer” means to significantly inhibittumor/cancer growth and/or metastasis. Growth inhibition can beindicated by reduced tumor volume or reduced occurrences of metastasis.Tumor growth can be determined, e.g., by examining the tumor volume viaroutine procedures (such as obtaining two-dimensional measurements witha dial caliper). Metastasis can be determined by inspecting for tumorcells in secondary sites or examining the metastatic potential ofbiopsied tumor cells in vitro using well-known techniques.

An “anti-nucleolin agent” includes any molecule, compound, etc., thatinteracts with nucleolin. Such agents include anti-nucleolin antibodiesand derivatives thereof, anti-sense oligonucleotides, ribozymes, RNAi,etc.

EMBODIMENTS

The following embodiments are given as examples of various ways topractice the invention. Many different ways of practicing the inventionare also possible.

In all embodiments, the underlying principle is to target cells fortherapeutic invention by specifically differentiating between plasmamembrane nucleolin and nuclear nucleolin. Plasma membrane nucleolin, asdiscovered by the applicants, correlates with cells that are in aneoplastic state. Exploiting this differential plasma membraneexpression, tumor and cancer cells can be targeted for treatment.

Detection of Plasma Membrane Nucleolin

Various techniques allow a user to differentiate between nuclear andplasma membrane nucleolin. Detection techniques, wherein thenucleolin-detecting reagents have exclusive access to extracellularportions of the cell (and consequently cell-plasma membrane nucleolin),or biochemical techniques, wherein either the surface plasma membraneand/or surface proteins are separated from other cellular components andcompartments, are also useful. One practicing the invention may wish todetermine the potential effectiveness of a therapy targeting nucleolinby first examining the cells for nucleolin plasma membrane expression.

In an embodiment, nucleolin is detected directly on the cell surface. Acell is isolated from a subject and plasma membrane nucleolin detectedusing an agent that binds nucleolin. Cells may be isolated by any knowntechnique. An isolated cell may comprise a larger tissue samplecontaining cells that are not neoplastic. Detection procedures useanti-nucleolin antibodies that bind extracellular nucleolin epitopes;these antibodies may be directly labeled or when bound, detectedindirectly. Other useful plasma membrane nucleolin detection agentsinclude GROs that specifically bind nucleolin. Useful procedures, suchas fluorescence-activated cell sorting (FACS) or immunofluorescence,employ fluorescent labels, while other cytological techniques, such ashistochemical, immunohistochemical and other microscopic (electronmicroscopy (EM), immuno-EM) techniques use various other labels, eithercolorimetric or radioactive. The various reagents may be assembled intokits.

In another embodiment, cells are isolated from a subject and extracted.Plasma membranes and/or proteins are then isolated (such as viadifferential extraction, or differential physical cell disruption,differential centrifugation of detergent-extracted cells, etc.), andthen nucleolin detected in the isolated membranes using an agent thatbinds nucleolin. In general, useful techniques to detect nucleolininclude those wherein the extract is placed on a substrate, and thesubstrate probed with a nucleolin-detecting reagent. Examples of suchtechniques include polypeptide dot blots and Western blots, biochips,protein arrays, etc. Other detection formats include enzyme-linkedimmunosorbent assays (ELISAs) in their manifold manifestations (Ausubelet al., 1987). In embodiments wherein plasma membrane surface moleculesare physically separated from most of the other cellular components andcompartments, the nucleolin-binding agents need not specificallyrecognize any extracellular portions of nucleolin. The various reagentsmay be assembled into kits.

In a further embodiment, the methods of the invention are directed todetecting lung cancer, such as lung small cell carcinomas. Plasmamembrane nucleolin expression is useful for detection and prognosis.

In one embodiment, the invention provides a method for theidentification of a subject for whom a certain therapy, such asnucleolin-directed chemotherapy, is indicated for the treatment oramelioration of a condition associated with an increased abundance ofnucleolin on the cell surface. Such a condition includes cancer,neoplasia and a precancerous lesion. Such a method may include the stepsof contacting an agent that binds specifically to nucleolin with a cellin or from a subject and determining the amount of binding of plasmamembrane nucleolin.

In another embodiment, an individual who has, in the past, presentedwith cancer presents to a health-care provider for examination. A samplehaving a cell, such as a biopsy specimen, is taken from the individual'sbody, whereupon the sample is contacted with an anti-nucleolin antibody,incubated, and the bound antibody then detected. The amount of boundantibody can be compared to the amount bound by healthy cells isolatedfrom the same individual. A treatment regimen may then be devised, basedon the quantity of cell-surface nucleolin per cell taken from theindividual's body and a known correlation between cell-surface nucleolinand susceptibility of the cancer to a certain manner of therapy, such aschemotherapy targeting nucleolin.

Treatment of Tumor/Cancer Cells Targeting Nucleolin

In one embodiment, methods of treating cells in a neoplastic stateincluding cancer and tumor cells, are provided; these methods exploitplasma membrane nucleolin which acts as a beacon for a therapeuticagent. For example, administration of anti-nucleolin antibodies, whichmay be conjugated to a toxin or other means of stimulating cell death orincurring necrosis, results in the removal of plasma membranenucleolin-expressing cells.

Practicing the Invention

The following, not meant to limit the invention, is presented to aid thepractitioner in carrying out the invention, although other methods,techniques, cells, reagents and approaches can be used to achieve theinvention.

Cells

Cells or tissue samples are collected from a subject. The subject is avertebrate, more preferably a mammal, such as a monkey, dog, cat,rabbit, cow, pig, goat, sheep, horse, rat, mouse, guinea pig, etc.; andmost preferably a human. Any technique to collect the desired cells maybe employed, including biopsy, surgery, scrape (inner cheek, skin, etc.)and blood withdrawal. Any appropriate tool may be used to carry out suchtasks. It is not necessary to isolate the test population (i.e., thosecells being tested for neoplastic state) from those cells and tissues(contaminating material) that are not being tested, except in some casesusing biochemical methods that include extraction. In this last case,the test population need not be completely isolated from contaminatingmaterials, but should either predominate or be easily distinguishable(e.g., morphologically (structurally, specific markers) orbiochemically).

For those methods that analyze lung carcinomas, sputum collection is anattractive and easily obtained sample. The term “sputum” as used hereinrefers to expectorated matter made up of saliva and discharges from therespiratory airways. Sputum is a highly complex material that has apronounced gel-like structure.

For collection of sputum, Byrne, et al., (Byrne, 1986) suggest that thepatient collect material, raised by several deep coughs, in a containerwith a lid. Alternatively, sputum can be collected by using abronchoscope (Kim et al., 1982). Specific devices or agents may be usedto facilitate sputum collection (Babkes et al., 2001; King and Speert,2002; Rubin and Newhouse, 1999). Other methods of sputum collection arealso available.

Cell Culture

In some cases, culturing the harvested cells is desirable to augmenttheir numbers so that plasma membrane nucleolin detection isfacilitated. Suitable media and conditions for generating primarycultures are well known. The selection of the media and cultureconditions vary depending on cell type and may be empiricallydetermined. For example, skeletal muscle, bone, neurons, skin, liver,and embryonic stem cells are grown in media that differs in theirspecific contents. Furthermore, media for one cell type may differsignificantly from laboratory to laboratory and institution toinstitution. To keep cells dividing, serum, such as fetal calf serum(FCS) (also known as fetal bovine serum (FBS)), is added to the mediumin relatively large quantities, 5%–30% by volume, depending on cell ortissue type. Other sera include newborn calf serum (NCS), bovine calfserum (BCS), adult bovine serum (ABS), horse serum (HS), human, chicken,goat, porcine, rabbit and sheep sera. Serum replacements may also beused, such as controlled process serum replacement-type (CPSR; 1 or 3)or bovine embryonic fluid. Specific purified growth factors or cocktailsof multiple growth factors can also be added or sometimes substitutedfor serum. Specific factors or hormones that promote proliferation orcell survival can also be used.

Examples of suitable culture media include Iscove's Modified Dulbecco'sMedium (IMDM), Dulbecco's Modified Eagle's Medium (DMEM), MinimalEssential Medium Eagle (MEM), Basal Medium Eagle (BME), Click's Medium,L-15 Medium Leibovitz, McCoy's 5A Medium, Glasgow Minimum EssentialMedium (GMEM), NCTC 109 Medium, Williams' Medium E, RPMI-1640, andMedium 199. A medium specifically developed for a particular celltype/line or cell function, e.g. Madin-Darby Bovine Kidney GrowthMedium, Madin-Darby Bovine Kidney Maintenance Medium, various hybridomamedia, Endothelial Basal Medium, Fibroblast Basal Medium, KeratinocyteBasal Medium, and Melanocyte Basal Medium are also known. If desired, aprotein-reduced or -free and/or serum-free medium and/or chemicallydefined, animal component-free medium may be used, e.g., CHO, GeneTherapy Medium or QBSF Serum-free Medium (Sigma Chemical Co.; St. Louis,Mo.), DMEM Nutrient Mixture F-12 Ham, MCDB (105, 110, 131, 151, 153, 201and 302), NCTC 135, Ultra DOMA PF or HL-1 (both from Biowhittaker;Walkersville, Md.), may be used.

The medium can be supplemented with a variety of growth factors,cytokines, serum, etc., depending on the cells being cultured. Examplesof suitable growth factors include: basic fibroblast growth factor(bFGF), vascular endothelial growth factor (VEGF), epidermal growthfactor (EGF), transforming growth factors (TGFα and TGFβ), plateletderived growth factors (PDGFs), hepatocyte growth factor (HGF),insulin-like growth factor (IGF), insulin, erythropoietin (EPO), andcolony stimulating factor (CSF). Examples of suitable hormone additivesare estrogen, progesterone, testosterone or glucocorticoids such asdexamethasone. Examples of cytokine medium additives are interferons,interleukins or tumor necrosis factor-α (TNFα). Salt solutions may alsobe added to the media, including Alseverr's Solution, Dulbecco'sPhosphate Buffered Saline (DPBS), Earle's Balanced Salt Solution, Gey'sBalanced Salt Solution (GBSS), Hanks' Balanced Salt Solution (HBSS),Puck's Saline A, and Tyrode's Salt Solution. If necessary, additives andculture components in different culture conditions be can optimized, asthese may alter cell response, activity lifetime or other featuresaffecting bioactivity. In addition, the surface on which the cells aregrown can be coated with a variety of substrates that contribute tosurvival, growth and/or differentiation of the cells. These substratesinclude but are not limited to, laminin, EHS-matrix, collagens,poly-L-lysine, poly-D-lysine, polyomithine and fibronectin. Whenthree-dimensional cultures are desired, extracellular matrix gels may beused, such as collagen, EHS-matrix, or gelatin (denatured collagen).Cells may be grown on top of such matrices, or may be cast within thegels themselves.

If desired, the media may be further supplemented with reagents thatlimit acidosis of the cultures, such as buffer addition to the medium(such as N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),bis(2-hydroxyethyl)amino -tris(hydroxymethyl)methane (BIS-Tris),N-(20hydroxyethyl)piperazine-N′3-propanesulfonic acid (EPPS or HEPPS),glyclclycine, N-2-hydroxyehtylpiperazine -N′-2-ethanesulfonic acid(HEPES), 3-(N-morpholino)propane sulfonic acid (MOPS),Piperazine-N,N′-bis(2-ethane-sulfonic acid) (PIPES), sodium bicarbonate,3-(N -tris(hydroxymethyl)-methyl-amino)-2-hydroxy-propanesulfonic acid)TAPSO, (N -tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), N-tris(hydroxymethyl)methyl-glycine (Tricine),tris(hydroxymethyl)-aminomethane (Tris), etc.). Frequent medium changesand changes in the supplied CO₂ (often approximately 5%) concentrationmay also be used to control acidosis.

Gases for culture typically are about 5% carbon dioxide and theremainder nitrogen, but optionally may contain varying mounts of nitricoxide (starting as low as 3 ppm), carbon monoxide and other gases, bothinert and biologically active. Carbon dioxide concentrations typicallyrange around 5%, but may vary between 2–10%. Both nitric oxide andcarbon monoxide, when necessary, are typically administered in verysmall amounts (i.e. in the pimp range), determined empirically or fromthe literature. The temperature at which the cells will grow optimallycan be empirically determined, although the culture temperature willusually be within the normal physiological range of the animal fromwhich the cells were isolated.

Detecting Nucleolin: Antibody-based Methods

Antibodies

Nucleolin can be detected at the protein level in cells, tissuesections, cultured cells and extracts thereof. Immunochemical methods todetect protein expression are well known and include, but are notlimited to, Western blotting, immunoaffinity purification,immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot orslot blotting, radioimmunoassay (RIA), immunohistochemical detection,immunocytochemical staining, and flow cytometry. Common procedures andinstructions using antibodies have been well addressed (e.g., (Harlowand Lane, 1988; Harlow and Lane, 1999). Selected antibodies that areuseful for detecting plasma membrane nucleolin are shown in Table 1.

TABLE 1 Anti-nucleolin antibodies Antigen Antibody Source source Notesp7-1A4 mouse Developmental Xenopus laevis IgG₁ monoclonal antibodyStudies Hybridoma oocytes (mAb) Bank (University of Iowa; Ames, IA)sc-8031 mouse mAb Santa Cruz Biotech human IgG₁ (Santa Cruz, CA) sc-9893goat Santa Cruz Biotech human IgG polyclonal Ab (pAb) sc-9892 goat pAbSanta Cruz Biotech human IgG clone 4E2 mouse MBL International humanIgG₁ mAb (Watertown, MA) clone 3G4B2 mouse Upstate dog IgG_(1k) mAbBiotechnology (Lake (MDCK cells) Placid, NY)

If additional anti-plasma membrane nucleolin antibodies are desired,they can be produced using well-known methods (Harlow and Lane, 1988;Harlow and Lane, 1999). For example, polyclonal antibodies (pAbs) can beraised in a mammalian host by one or more injections of an immunogen,such as an extracellular domain of surface-expressed nucleolin, and, ifdesired, an adjuvant. Typically, the immunogen (and adjuvant) isinjected in a mammal by a subcutaneous or intraperitoneal injection. Theimmunogen may include components such as polypeptides (isolated,non-isolated, or recombinantly produced), cells or cell fractions.Examples of adjuvants include Freund's complete, monophosphoryl Lipid Asynthetic-trehalose dicorynomycolate, aluminum hydroxide (alum), heatshock proteins HSP 70 or HSP96 (WO01/917871A1), squalene emulsioncontaining monophosphoryl lipid A (LaPosta and Eldrige, 2001),a₂-macroglobulin and surface active substances, including oil emulsions,pleuronic polyols, polyanions and dinitrophenol. To improve the immuneresponse, an immunogen may be conjugated to a polypeptide that isimmunogenic in the host, such as keyhole limpet hemocyanin (KLH), serumalbumin, bovine thyroglobulin, cholera toxin, labile enterotoxin, orsoybean trypsin inhibitor. Alternatively, pAbs may be made in chickens,producing IgY molecules (Schade et. al., 1996).

Monoclonal antibodies (mAbs) may also be made by immunizing a host orlymphocytes from a host, harvesting the mAb-secreting (or potentiallysecreting) lymphocytes, fusing those lymphocytes to immortalized cells(e.g., myeloma cells), and selecting those cells that secrete thedesired mAb (Goding, 1996; Kohler and Milstein, 1975). Other techniquesmay be used, such as EBV-hybridoma technique (Cole et al., 1985;Coligan, 1996). Techniques for the generation of chimeric antibodies bysplicing genes encoding the variable domains of non-human antibodies togenes of the constant domains of human immunoglobulin result in“chimeric antibodies” that are substantially human at the amino acidlevel (Neuberger, Williams et al. 1984; Morrison, Johnson et al. 1984).If desired, the mAbs may be purified from the culture medium or ascitesfluid by conventional procedures, such as protein A-sepharose,hydroxylapatite chromatography, gel electrophoresis, dialysis, ammoniumsulfate precipitation or affinity chromatography (Harlow and Lane, 1988;Harlow and Lane, 1999). Additionally, human monoclonal antibodies can begenerated by immunization of transgenic mice containing a third copy IgGhuman trans-loci and silenced endogenous mouse Ig loci (Surani et al.,1996) or using human-transgenic mice (Jakobovits et al., 1998; Lonbergand Kay, 1998). Production of humanized monclonal antibodies andfragments thereof can also be generated through phage displaytechnologies (Winter, Griffiths et al. 1994).

An example of the production of a murine monoclonal antibody to humannucleolin according to described techniques (Kohler and Milstein, 1975)is as follows. Female BALB/c mice (20–25 g) are injectedintraperitoneally with 100 μg of antigen containing human nucleolinpolypeptide, or portion thereof. Alternatively, the antigen includesmurine cells which have been transformed to express human nucleolin.After 2 weeks, a second injection having 50 μg of antigen is injected.To test for production of anti-nucleolin antibodies, sera from the miceare used in immunohistologic screening. Mice displaying high blood serumlevels of anti-nucleolin antibody receive a third injection (20 μg) ofantigen. Four days later, the mice are sacrificed, their spleen cellsisolated and fused with a myeloma line, e.g., P3X63Ag8.653 (AmericanType Tissue Collection; Manassas, Va.). The resulting hybridoma cellsare cultured, sub-cloned and selected for expression of antibodieshaving high affinities for nucleolin.

Non-immunogenic human-like or humanized polyclonal antibodies that bindto nucleolin may also be produced. These polyclonal antibodies can bemade, for example, using phage display methods (Sharon, 1995), or byimmunizing transgenic or genetically engineered animals capable ofproducing human polyclonal antibodies (Singh and Dias, 2002).

Antibodies Suitable for Therapy

Antibodies most ideal for use as therapeutics are those that arenon-immunogenic when administered to subjects. Such antibodies have theadvantages of exerting minimal side-effects, having long serum andbiologic half-life, having wide bio-distribution, having high targetspecificity and high activity in engaging the effector phase of theimmune system. These antibodies, when intended for human subjects, arecommonly referred to as “humanized,” “human,” “chimeric,” or“primatized” antibodies; these are comprised substantially (>70%) ofhuman amino acid sequences.

Detection

An approach using antibodies to detect the presence of an antigen willinclude one or more, if not all, of the following steps:

(1) Preparing the entity being tested for plasma membrane nucleolin bywashing with buffer or water

(2) Blocking non-specific antibody binding sites

(3) Applying the antibody (e.g., nucleolin)

(4) Detecting bound antibody, either via a detectable labeled-secondaryantibody that recognizes the primary antibody or a detectable label thathas been directly attached to, or associated with, the bound(anti-nucleolin) antibody.

Substrates may be washed with any solution that does not interfere withthe epitope structure. Common buffers include saline and biologicalbuffers, such as bicine, tricine, and Tris.

Non-specific binding sites are blocked by applying a protein solution,such as bovine serum albumin (BSA; denatured or native), milk proteins,or in the cases wherein the detecting reagent is a secondary antibody,normal serum or immunoglobulins from a non-immunized host animal whosespecies is the same origin as the detecting antibody. For example, aprocedure using a secondary antibody made in goats would employ normalgoat serum (NGS).

The substrate is then reacted with the antibody of interest. Theantibody may be applied in any form, such as F_(ab) fragments andderivatives thereof, purified antibody (by affinity, precipitation,etc.), supernatant from hybridoma cultures, ascites, serum orrecombinant antibodies expressed in recombinant cells. The antibody maybe diluted in buffer or media, often with a protein carrier such as thesolution used to block non-specific binding sites; the useful antibodyconcentration is usually determined empirically. In general, polyclonalsera, purified antibodies and ascites may be diluted 1:50 to 1:200,000,more often, 1:200 to 1:500. Hybridoma supernatants may be diluted 1:0 to1:10, or may be concentrated by dialysis or ammonium sulfateprecipitation (or any other method that retains the antibodies ofinterest but at least partially removes the liquid component andpreferably other small molecules, such as salts) and diluted ifnecessary. Incubation with antibodies may be carried out for as littleas 20 minutes at 37° C., 2 to 6 hours at room temperature (approximately22° C.), or 8 hours or more at 4° C.

To detect an antibody-antigen complex, a label may be used. The labelmay be coupled to the binding antibody, or to a second antibody thatrecognizes the first antibody, and is incubated with the sample afterthe primary antibody incubation and thorough washing. Suitable labelsinclude fluorescent moieties, such as fluorescein isothiocyanate;fluorescein dichlorotriazine and fluorinated analogs of fluorescein;naphthofluorescein carboxylic acid and its succinimidyl ester;carboxyrhodamine 6G; pyridyloxazole derivatives; Cy2, 3 and 5;phycoerythrin; fluorescent species of succinimidyl esters, carboxylicacids, isothiocyanates, sulfonyl chlorides, and dansyl chlorides,including propionic acid succinimidyl esters, and pentanoic acidsuccinimidyl esters; succinimidyl esters of carboxytetramethylrhodamine;rhodamine Red-X succinimidyl ester; Texas Red sulfonyl chloride; TexasRed-X succinimidyl ester; Texas Red-X sodium tetrafluorophenol ester;Red-X; Texas Red dyes; tetramethylrhodamine; lissamine rhodamine B;tetramethylrhodamine; tetramethylrhodamine isothiocyanate;naphthofluoresceins; coumarin derivatives; pyrenes; pyridyloxazolederivatives; dapoxyl dyes; Cascade Blue and Yellow dyes; benzofuranisothiocyanates; sodium tetrafluorophenols;4,4-difluoro-4-bora-3a,4a-diaza-s-indacene. Suitable labels furtherinclude enzymatic moieties, such as alkaline phosphatase or horseradishperoxidase; radioactive moieties, including ³⁵S and ¹³⁵I-labels; avidin(or streptavidin)-biotin-based detection systems (often coupled withenzymatic or gold signal systems); and gold particles. In the case ofenzymatic-based detection systems, the enzyme is reacted with anappropriate substrate, such as 3,3′-diaminobenzidine (DAB) forhorseradish peroxidase; preferably, the reaction products are insoluble.Gold-labeled samples, if not prepared for ultrastructural analyses, maybe chemically reacted to enhance the gold signal; this approach isespecially desirable for light microscopy. The choice of the labeldepends on the application, the desired resolution and the desiredobservation methods. For fluorescent labels, the fluorophore is excitedwith the appropriate wavelength and the sample observed using amicroscope, confocal microscope, or FACS machine. In the case ofradioactive labeling, the samples are contacted with autoradiographyfilm, and the film developed; alternatively, autoradiography may also beaccomplished using ultrastructural approaches. Alternatively,radioactivity may be quantified using a scintillation counter.

The invention also provides methods for the detection of a tumor in anindividual in vivo. The individual is administered a pharmaceuticallyacceptable composition having an agent that binds cell-surfacenucleolin. The agent also incorporates a detectable label, such as aradiolabel, which distribution in the subject's body is then mapped. Theagent may comprise an aptamer, an oligonucleotide, a peptide, a smallmolecule, or a macromolecule, such as an antibody.

In one example of the method, a composition that specifically binds totumor cell surface nucleolin is radiolabeled. The nature of theradiolabel is determined by the device that is used to record thepresence and distribution of the agent in the patient's body. Forexample, if standard radio-imaging using gamma cameras is used, any oneof several radioisotopes is used, such as technetium-99 or indium-111.Alternatively, if positron emission tomography (PET) is used, then theradiolabel is a radioactive halogen, such as flourine-18. The patient isinjected intravenously with the radiolabeled agent, and scans of thepatient, to visualize the location of the agent, are performed over thefollowing 12 hour period, at, for example, 1, 4, and 12 hourspost-injection. Monitoring techniques include whole body scanning, SPECT(which allows cross-sections of the body to be visualized), and anytechnique that permits monitoring of emission.

Cytological-based Approaches:

Immunofluorescence/immunohistochemical

Protein expression by cells or tissue can be ascertained byimmunolocalization of an antigen. Generally, cells or tissue arepreserved by fixation, exposed to an antibody that recognizes theepitope of interest, such as a nucleolin, and the bound antibodyvisualized.

Any cell, cell line, tissue, or even an entire organism is appropriatefor fixation. Cells may be cultured in vitro as primary cultures, celllines, or harvested from tissue and separated mechanically orenzymatically. Tissue may be from any organ, plant or animal, and may beharvested after or prior to fixation. Fixation, if desired, may be byany known means; the requirements are that the protein to be detected benot rendered unrecognizable by the binding agent, most often anantibody. Appropriate fixatives includeparaformaldehyde-lysine-periodate, formalin, paraformaldehyde, methanol,acetic acid-methanol, glutaraldehyde, acetone, Karnovsky's fixative,etc. The choice of fixative depends on variables such as the protein ofinterest, the properties of a particular detecting reagent (such as anantibody), the method of detection (fluorescence, enzymatic) and themethod of observation (epifluorescence microscopy, confocal microscopy,light microscopy, electron microscopy, etc.). The sample is usuallyfirst washed, most often with a biological buffer, prior to fixation.Fixatives are prepared in solution or in biological buffers; manyfixatives are prepared immediately prior to applying to the sample.Suitable biological buffers include saline (e.g., phosphate bufferedsaline), N -(carbamoylmethyl)-2-aminoethanesulfonic acid (ACES),N-2-acetamido-2-iminodiacetic acid (ADA), bicine, bis-tris,3-cyclohexylamino-2-hydroxy-1-propanesulfonic acid (CAPSO),ethanolamines, glycine, N-2-hydroxyethylpiperazine -N′-2-ethanesulfonicacid (HEPES), 2-N-morpholinoethanesulfonic acid (MES), 3-N-morpholinopropanesulfonic acid (MOPS), 3-N-morpholino-2-hyrdoxy-propanesulfonic acid (MOPSO), piperazine-N,N′-bis(2-ethanesulfonicacid) (PIPES), tricine, triethanolamine, etc. An appropriate buffer isselected according to the sample being analyzed, appropriate pH, and therequirements of the detection method. A useful buffer is phosphatebuffered saline (PBS). After fixation, the sample may be stored infixative, preferably fresh, or temporarily or indefinitely, at atemperature between about 4° C. to about 22° C.

After fixation from 5 minutes to 1 week, depending on the sample size,sample thickness, and viscosity of the fixative, the sample is washed inbuffer. If the sample is thick or sections are desired, the sample maybe embedded in a suitable matrix. For cryosectioning, sucrose isinfused, and embedded in a matrix, such as OCT Tissue Tek (AndwinScientific; Canoga Park, Calif.) or gelatin. Samples may also beembedded in paraffin wax, or resins suitable for electron microscopy,such as epoxy-based (Araldite, Polybed 812, Durcupan ACM, Quetol,Spurr's, or mixtures thereof, Polysciences, Warrington, Pa.), acrylates(London Resins (LR White, LR gold), Lowicryls, Unicryl; Polysciences),methylacrylates (JB-4, OsteoBed; Polysciences), melamine (Nanoplast;Polysciences) and other media, such as DGD, Immuno-Bed (Polysciences)and then polymerized. Resins that are especially appropriate includehydrophilic (such as Lowicryls, London Resins, water-soluble Durcupan,etc.) since these are less likely to denature the protein of interestduring polymerization and will not repel antibody solutions. Whenembedded in wax or resin, samples are dehydrated by passing them througha concentration series of ethanol or methanol; in some cases, othersolvents may be used, such as polypropylene oxide. Embedding may occurafter the sample has been reacted with the detecting agents, or samplesmay be first embedded, sectioned (via microtome, cyrotome, orultramicrotome), and then the sections reacted with the detectingreagents. In some cases, the embedding material may be partially orcompletely removed before detection to facilitate antigen access.

In some instances, the nucleolin epitope(s) to which the antibody bindsmay be rendered unavailable because of fixation. Antigen retrievalmethods can be used to make the antigen available for antibody binding.Many recourses are available (reviewed in, for example, (McNicol andRichmond, 1998; Robinson and Vandre, 2001; Shi et al., 2001)). Commonmethods include using heat supplied from autoclaves, microwaves, hotwater or buffers, pressure cookers, or other sources of heat. Often thesources of heat are used in sequence; the samples must often be insolution (e.g., microwave treatments). Detergent treatment may alsounmask antigens, such as sodium dodecyl sulfate (SDS, 0.25% to 1%) orother denaturing detergents. Chemical methods include strong alkalis(such as NaOH), prolonged immersion in water, urea, formic acid andrefixation in zinc sulfate-formalin. In other instances, proteolyticenzyme treatment will modify the antigen such that it is available tothe antibody. Any number of proteases may be used, such as trypsin.These methods may be combined to achieve optimal results. The choice ofthe antigen retrieval method will depend on the sample, its embedment(if any), and the anti-nucleolin antibody.

Especially in the cases of immunofluorescent or enzymatic product-baseddetection, background signal due to residual fixative, proteincross-linking, protein precipitation or endogenous enzymes may bequenched, using, e.g., ammonium chloride or sodium borohydride or asubstance to deactivate or deplete confounding endogenous enzymes, suchas hydrogen peroxide which acts on peroxidases. To detect intracellularproteins in samples that are not to be sectioned, samples may bepermeabilized. Permeabilizing agents include detergents, such ast-octylphenoxypolyethoxyethanols, polyoxyethylenesorbitans, and otheragents, such as lysins, proteases, etc.

Non-specific binding sites are blocked by applying a protein solution,such as bovine serum albumin (BSA; denatured or native), milk proteins,or preferably in the cases wherein the detecting reagent is an antibody,normal serum or IgG from a non-immunized host animal whose species isthe same is the same origin of the detecting antibody.

Flow Cytometry/Fluorescence-Activated Cell Sorting (FACS)

Methods of performing flow cytometry are well known (Orfao andRuiz-Arguelles, 1996). Because plasma membrane nucleolin is beingprobed, cell permeabilization that allows access to cytoplasmiccompartments is undesirable. After harvesting, cells are prepared as asingle-cell suspension; cells are then incubated with an anti-nucleolinantibody usually after blocking non-specific binding sites. Preferably,the anti-nucleolin antibody is labeled with a fluorescent marker. If theantibody is not labeled with a fluorescent marker, a second antibodythat is immunoreactive with the first antibody and contains afluorescent marker is used. After sufficient washing to ensure thatexcess or unbound antibodies are removed, the cells are ready for flowcytometry.

Biochemical-based Approaches:

In these approaches, it is first desirable to isolate plasma membraneproteins from other cellular compartments. This may be done in anynumber of ways, such as simple cell extraction, differential extractionor mechanical disruption followed by separation of cellular compartmentson gradients (such as sucrose or polydextran) by centrifugation,extraction followed by immunoselecting appropriate cellular compartmentswith plasma membrane-specific antibodies, etc. An example of such anapproach is described in (Naito et al., 1988; Yao et al., 1996b)).Extracting reagents are well known. For examples, solvents such asmethanol may be occasionally useful. More likely, detergents, such ast-octylphenoxypolyethoxyethanol (also known as polyethylene glycoltert-octylphenyl ether) are particularly useful for simple extractions.Also useful are glucopyranosides, maltopyranosides, maltosides,polyoxyethylene esters, other polyoxyethylene ethers, salts of alginic,caprylic, cholic 1-decanesulfonic, deoxycholic, dioctyl sulfosuccinate,1-dodecanesulfonic, glyocholic, glycodeoxycholic, 1-heptanesulfonic,1-hexanesulfonic, N-lauroylsacrosine, lauryl sulfate (e.g., SDS),1-nonanesulfonic, 1-octanesulfonic, 1-pentanesulfonic, taurocholic andtauodexycholic acids; sodium 7-ethyl-2-methyl-4-undecyl sulfate, andsodium 2-ethylhexyl sulfate. Other useful detergentsinclude(3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate,(3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane-sulfonate,N-decyl-, N -dodecyl-, N-hexadecyl-, N-octadecyl-,N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonates andphosphatidylcholine. Less useful, but may be helpful in some cases, arealkyltrimethylammonium bromides, benzalkonium chloride, benzethoniumchloride, benzyldimethyldodecylammonium bromide,benzyldimethylhexadecylammonium chloride, cetyldimethylethylammoniumbromide, cetylpyridinium, decamethonium bormide,dimethyldioctadecylammonium bromide, methylbenzethonium chloride,methyltiroctylammonium chloride, andN,N′,N′-polyoxyehtlylene(10)-N-tallow-1,3-diaminopropane. The differentextracting reagents may be used singly or in combination; they may beprepared in simple aqueous solutions or suitable buffers.

Polyethylene glycol ter-octylphenyl ether is particularly useful fordifferential extraction by taking advantage of the low cloud point toseparate membrane proteins from soluble proteins into two differentphases.

Extraction buffers may contain protease inhibitors, such as aprotinin,benzamidine, antipain, pepstatin and iodoacetamide.

Extracts are then assayed for nucleolin expression. For those techniquesthat separate surface plasma membrane from other cellular components(especially the nucleus), the nucleolin detecting agents need not bespecific for extracellular plasma membrane nucleolin epitopes.

Immunosorbent Assay (ELISA) (Ausubel et al., 1987)

Various types of enzyme linked immunosorbent assays (ELISAs) to detectprotein expression are known, and these are applicable to nucleolindetection. However, other ELISA-like assays include radio-immunoassaysand other non-enzyme linked antibody binding assays and procedures. Inthese assays, the cell surface proteins are the principle components inthe cell preparation.

The double antibody-sandwich ELISA technique is especially useful. Thebasic protocol for a double antibody-sandwich ELISA is as follows: Aplate is coated with anti-nucleolin antibodies (capture antibodies). Theplate is then washed with a blocking agent, such as BSA, to blocknon-specific binding of proteins (antibodies or antigens) to the testplate. The test sample is then incubated on the plate coated with thecapture antibodies. The plate is then washed, incubated withanti-nucleolin antibodies, washed again, and incubated with a specificantibody-labeled conjugates and the signal appropriately detected.

In other ELISAs, proteins or peptides are immobilized onto a selectedsurface, the surface exhibit may have affinity for proteins, such as thewells of a specially-treated polystyrene microtiter plate. After washingto remove incompletely adsorbed material, one would then generallydesire to bind or coat with a nonspecific protein that is known to beantigenically neutral with anti-nucleolin antibodies, such as BSA orcasein, onto the well bottom. This step allows for blocking ofnonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific binding of antibodies ontothe surface. When the antibodies were created in an animal byconjugating a polypeptide to a protein (e.g., BSA), a different proteinis usually used as a blocking agent, because of the possibility of thepresence of antibodies to the blocking protein the antibody composition.

After binding of nucleolin to the well, coating with a non-reactivematerial to reduce background, and washing to remove unbound material,the immobilizing surface is contacted with an anti-nucleolin antibodycomposition in a manner conducive to immune complex (antigen/antibody)formation. Such conditions include diluting the antibody compositionwith diluents such as BSA, bovine γ globulin (BGG) andPBS/Polyoxyethylenesorbitan monolaurate. These added agents also assistin the reduction of nonspecific background signal. The layered antibodycomposition is then allowed to incubate for, e.g., from 2 to 4 hours at25° C. to 37° C. Following incubation, the antibodycomposition-contacted surface is washed so as to removenon-immunocomplexed material. One washing procedure includes washingwith a PBS/polyoxyethylenesorbitan monolaurate or borate buffersolution.

Following formation of specific immunocomplexes between the test sampleand the antibody and subsequent washing, immunocomplex formation isdetected using a second antibody having specificity for theanti-nucleolin antibody. For detection, the secondary antibody isassociated with detectable label, such as an enzyme or a fluorescentmolecule. A number of immunoassays are discussed in U.S. Pat. Nos.5,736,348, 5,192,660, and 4,474,892.

Western Blotting (Ausubel et al., 1987)

Western blotting methods are well known. Generally, a protein sample issubjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) at such conditions as to yield an appropriate separation ofproteins within the sample. The proteins are then transferred to amembrane (e.g., nitrocellulose, nylon, etc.) in such a way as tomaintain the relative positions of the proteins to each other.

Visibly labeled proteins of known molecular weight may be includedwithin a lane of the gel. These proteins serve as a control to insureadequate transfer of the proteins to the membrane, as well as molecularweight markers for determining the relative molecular weight of otherproteins on the blot. Alternatively, unlabeled marker proteins (or insome rare instances, no marker proteins) are detected after transferwith Brilliant Blue (G or R; Sigma; St. Louis, Mo.) other protein dyes.After protein transfer, the membrane is submersed in a blocking solutionto prevent nonspecific binding of the primary antibody.

The primary antibody, e.g., anti-nucleolin, may be labeled and thepresence and molecular weight of the antigen may be determined bydetecting the label at a specific location on the membrane. However, theprimary antibody may not be labeled, and the blot is further reactedwith a labeled second antibody. This secondary antibody isimmunoreactive with the primary antibody; for example, the secondaryantibody may be one to rabbit imunoglobulins and labeled with alkalinephosphatase. An apparatus for and methods of performing Western blotsare described in U.S. Pat. No. 5,567,595.

Immunoprecipitation (Ausubel et al. 1987: Harlow and Lane, 1999)

Protein expression can be determined and quantified by isolatingantigens using immunoprecipitation. Methods of immunoprecipitations aredescribed in U.S. Pat. No. 5,629,197. Immunoprecipitation involves theseparation of the target antigen component from a complex mixture and isused to discriminate or isolate minute amounts of protein. For theisolation of cell-surface proteins, nonionic salts are often used.

For example, an immunoprecipitation from whole cells may be performed asfollows. Cells are extracted with one or more detergents (see above),such as, for example, 1% t-octylphenoxypolyethoxyethanol/0.1% SDS/150 mMNaCl in 20 mM Tris buffer, pH 8.6. After extraction, which may be aidedby agitation, insoluble debris is removed using a centrifuge.Anti-nucleolin antibody is added to the extracts, and then the samplesare incubated 30 minutes to overnight at 4° C. Staphylococcus aureus orrecombinantly-produced Protein A or Group C Staphylococcus Protein Gconjugated to sepharose or tris-acryl beads are then added. In thoseinstances when the anti-nucleolin antibody does not bind well to ProteinA, IgG Abs that recognize antibodies of the animal in which theanti-nucleolin antibody was made is simultaneously added. The samplesare then incubated with gentle agitation for around 2 hours at 4° C. Thebeads or bacterial cells, now bound to the antibody-antigen complexes,are thoroughly washed, usually first with either the extraction solutionor a high salt buffer, then a salt-less buffer or water to removenonspecifically-bound proteins and residual detergent molecules. Afterremoving residual buffer, the beads are incubated with a buffer, such aselectrophoresis sample buffer, and then subjected to 95° C. for 3–5minutes to elute bound proteins from the beads. The samples are thenready for analysis and nucleolin detection.

Other Methods:

Immunoselection Procedures (Other than FACS) (Ausubel et al., 1987)

Cells expressing plasma membrane nucleolin can be easily isolated by“panning” on plastic plates coated with anti-antibody antibodies(Wysocki and Sato, 1978). Panning has many advantages over otherimmunoselection procedures: It is fast, efficient (10⁷ cells can easilybe panned on two 60-mm plastic plates in 30 minutes), and inexpensive.

In general, a single cell suspension is labeled with an anti-nucleolinantibody, and then is incubated on a substrate coated with a secondaryantibody (with non-specific binding sites blocked). After 1 to 3 hoursincubation at room temperature, non-adherent cells are washed away. Inthis embodiment, bound cells indicate that nucleolin is expressed in theplasma membrane, indicating a neoplastic cell.

Detecting Nucleolin: Oligonucleotide-based Methods

GROs and other oligonucleotides that recognize and bind nucleolin (Bateset al., 1999; Miller et al., 2000; Xu et al., 2001) can be used much thesame way as antibodies are. Examples of suitable assays are given below.In some cases, incorporating the GRO nucleotides into larger nucleicacid sequences may be advantageous; for example, to facilitate bindingof a GRO nucleic acid to a substrate without denaturing thenucleolin-binding site.

Useful GROs that bind nucleolin (and also have the biological propertyof inhibiting cancer cell growth) have been described (Bates et al.,1999; Miller et al., 2000; Xu et al., 2001). They include those shown inTable 2. Control GROs are useful for detecting background signal levels.

TABLE 2 Non-anti-sense GRO that bind nucleolin and non-bindingcontrols^(1,2,3) SEQ GRO Sequence ID NO: GRO29A¹ tttggtggtg gtggttgtggtggtggtgg  1 GRO29-2 tttggtggtg gtggttttgg tggtggtgg  2 GRO29-3tttggtggtg gtggtggtgg tggtggtgg  3 GRO29-5 tttggtggtg gtggtttgggtggtggtgg  4 GRO29-13 tggtggtggt ggt  5 GRO14C ggtggttgtg gtgg  6 GRO15Agttgtttggg gtggt  7 GRO15B² ttgggggggg tgggt  8 GRO25A ggttggggtgggtggggtgg gtggg  9 GRO26B¹ ggtggtggtg gttgtggtgg tggtgg 10 GRO28Atttggtggtg gtggttgtgg tggtggtg 11 GRO28B tttggtggtg gtggtgtggt ggtggtgg12 GRO29-6 ggtggtggtg gttgtggtgg tggtggttt 13 GRO32A ggtggttgtggtggttgtgg tggttgtggt gg 14 GRO32B tttggtggtg gtggttgtgg tggtggtggt tt15 GRO56A ggtggtggtg gttgtggtgg tggtggttgt 16 ggtggtggtg gttgtggtggtggtgg CRO tttcctcctc ctccttctcc tcctcctcc 18 GRO A ttagggttagggttagggtt aggg 19 GRO B ggtggtggtg g 20 GRO C ggtggttgtg gtgg 21 GRO Dggttggtgtg gttgg 22 GRO E gggttttggg 23 GRO F ggttttggtt ttggttttgg 24GRO G¹ ggttggtgtg gttgg 25 GRO H¹ ggggttttgg gg 26 GRO I¹ gggttttggg 27GRO J¹ ggggttttgg ggttttgggg ttttgggg 28 GRO K¹ ttggggttgg ggttggggttgggg 29 GRO L¹ gggtgggtgg gtgggt 30 GRO M¹ ggttttggtt ttggttttgg ttttgg31 GRO N² tttcctcctc ctccttctcc tcctcctcc 32 GRO O² cctcctcctccttctcctcc tcctcc 33 GRO P² tggggt 34 GRO Q² gcatgct 35 GRO R²gcggtttgcg g 36 GRO S² tagg 37 GRO T² ggggttgggg tgtggggttg ggg 38¹Indicates a good plasma membrane nucleolin-binding GRO. ²Indicates anucleolin control (non-plasma membrane nucleolin binding). ³GRO sequencewithout ¹ or ² designations have some anti-proliferative activity.Cytological-based Approaches:Cellular Localization/labeling (Relative of Immuno-basedLocalization/labeling Assays)

The procedures outlined above for the immuno-based localization assays(such as immunofluorescence or FACS) are also applicable to those assayswherein the detecting reagent is a nucleolin-binding GRO. Modificationsinclude those to prevent non-specific binding, using denatured DNA, suchas from salmon sperm instead of a protein such as BSA. For detection,similar labels as outlined above are also useful as long as the GRO canbe derivatized with the label in some form. For this purpose,biotin-avidin nucleic acid labeling systems are especially convenient,as are digoxigenin ones (Ausubel et al., 1987). The synthesis ofbiotinylated nucleotides has been described (Langer et al., 1981).Biotin, a water-soluble vitamin, can covalently attached to the C5position of the pyrimidine ring via an alylamine linker arm; biotinnon-covalently binds avidin or streptavidin, which may be easilylabeled. Alternatively, biotin is added to oligonucleotides duringsynthesis by coupling to the 5′-hydroxyl of the terminal nucleotide.Digoxigenin-11-dUTP can be incorporated into DNA by either nicktranslation or random oligonucleotide-primed synthesis protocols.Digoxigenin is detected using labeled anti-digoxigenin antibodies.Convenient digoxigenin systems are commercially available (RocheMolecular Biochemicals; Indianapolis, Ind.). An example of a procedureusing oligonucleotides to detect and localize proteins has beendescribed by (Davis et al., 1998).

Biochemical-based Approaches:

GROs may also be used in a similar fashion as antibodies to detectnucleolin in biochemical approaches, as described above. For example,“Southwestern”-type blotting experiments may be performed with GROs(Bates et al., 1999; Miller et al., 2000). After cells have beenappropriately extracted (for example, differentially to separate plasmamembrane proteins from intracellular proteins), the proteins aresubjected to electrophoresis on polyacrylamide gels and transferred to asubstrate, such as a polyvinliden difluoride membrane. Proteins aredenatured and renatured by washing for 30 minutes at 4° C. with 6 Mgaunidine-HCl, followed by washes in 3 M, 1.5 M and 0.75 M guanidine HClin 25 mM HERPES (pH 7.9)/4 mM KCl/3 mM MgCl₂). After blockingnon-specific binding sites with 5% non-fat dried milk in HEPES buffer,the labeled GRO is hybridized for 2 hours at 4° C. in HEPES bindingbuffer supplemented with 0.25% NDM, 0.05% NP-40, 400 ng/ml salmon spermDNA and 100 ng/ml of an unrelated mixed sequence oligonucleotide, suchas tcgagaaaaa ctctcctctc cttccttcct ctcca; SEQ ID NO:17. After washingwith HEPES binding buffer, the signal is detected appropriately.

Other Methods:

Arrays

Arrays of Immobilized Nucleolin-binding Reagents on Chips

A chip is an array of regions containing immobilized molecules,separated by regions containing no molecules or immobilized molecules ata much lower density. For example, a protein chip may be prepared byapplying nucleolin-binding antibodies; an “aptamer”-like chip may beprepared by applying nucleolin binding GROs. The remaining regions areleft uncovered or are covered with inert molecules. The arrays can berinsed to remove all but the specifically immobilized polypeptides ornucleic acids. In addition, chips may also be prepared containingmultiple nucleolin-binding antibodies (Table 1), nucleic acids (such asGROs; Table 2), or both, and may contain control antibodies and/ornucleic acids that are non-reactive with nucleolin. Such an array wouldallow for simultaneous test confirmation, duplication and internalcontrols.

Proteins, such as anti-nucleolin antibodies, can be immobilized ontosolid supports by simple chemical reactions, including the condensationof amines with carboxylic acids and the formation of disulfides. Thiscovalent immobilization of proteins on inert substrates can prevent highbackground signals due to non-specific adsorption. Substratesderivatized with other molecules, such as biotin, are also useful whenthe protein to be immobilized is derivatized with avidin orstreptavidin, or vice-versa. In some rare cases, especially whenanti-nucleolin antibody-encoding nucleic acid sequences are available,fusion polypeptides comprising anti-nucleolin antibody may beadvantageous for immobilization onto a substrate.

The surface may be any material to which a the nucleolin binding agentcan be immobilized. For example, the surface may be metal, glass,ceramic, polymer, wood or biological tissue. The surface may include asubstrate of a given material and a layer or layers of another materialon a portion or the entire surface of the substrate. The surfaces may beany of the common surfaces used for affinity chromatography, such asthose used for immobilization of glutathione for the purification of GSTfusion polypeptides. The surfaces for affinity chromatography include,for example, sepharose, agarose, polyacrylamide, polystyrene anddextran. The surface need not be a solid, but may be a colloid, anexfoliated mineral clay, a lipid monolayer, a lipid bilayer, a gel, or aporous material.

The immobilization method desirably controls the position of thenucleolin binding agent on the surface; for example, enabling theantigen binding portions of antibodies unattached to the substrate,while the non-antigen binding portions are rooted to the substrate. Bycontrolling the position of individual reactant ligands, patterns orarrays of the ligands may be produced. The portions of the surface thatare not occupied by the nucleolin-binding reagent do not allownon-specific adsorption of polypeptides or polynucleotides.

In this embodiment, a sample from a subject, for example, blood, ispassed over a chip containing nucleolin-binding molecules. A biosensingdevice, such as machine that detects changes in surface plasmonresonance, is then used to detect bound nucleolin. BIAcore (Uppsala,Sweden) chips serve as examples of useful chips and detection machines.

Prognostic Assays

Diagnostic methods can furthermore be used to identify subjects having,or at risk of developing, a neoplasia at an early stage of diseasedevelopment, since the surface expression of nucleolin can be detectedearlier than in conventional methods. Prognostic assays can be used toidentify a subject having or at risk for developing a neoplasia, such asa subject who has a family history of harmful neoplasias, especiallycancers. A method for identifying such an individual would include atest sample obtained from a subject and testing for cell surfacelocalization of nucleolin.

In another embodiment, detecting plasma membrane nucleolin and theneither qualitatively or quantitatively assessing the amount of nucleolin(usually indirectly through the signal generated from bound nucleolinmolecules) can indicate the rate of cell proliferation, since plasmamembrane nucleolin levels correlate with cell proliferation rates.

Kits

Kits, containers, packs, or dispensers containing nucleolin probes anddetection reagents, together with instructions for administration, maybe assembled. When supplied as a kit, the different components may bepackaged in separate containers and admixed immediately before use. Suchpackaging of the components separately may permit long-term storagewithout losing the active components' functions.

Kits may also include reagents in separate containers that facilitatethe execution of a specific test, such as diagnostic tests. For example,non-nucleolin-binding GROs may be supplied for internal negativecontrols, or nucleolin and a nucleolin-binding reagent for internalpositive controls. The components of a kit are an anti-nucleolin agentused to probe for nucleolin, a control sample, and optionally acomposition to detect nucleolin. Examples of anti-nucleolin agentsinclude an anti-nucleolin antibody (e.g., as shown in Table 1) orfragment thereof; if labeled, then a nucleolin-binding detection reagentis superfluous. A nucleolin-binding oligonucleotide (e.g., as shown inTable 2), which may be derivatized such that a second labeled reagentmay bind (such as biotin). However, if a labeled GRO nucleic acid isprovided, then a second labeled reagent is superfluous. Examples ofdetection reagents include: labeled secondary antibodies, for example,an anti-mouse pAb made in donkey and then tagged with a fluorophore suchas rhodamine, or a labeled reagent to detect oligonucleotides such asGROs; for example, avidin or streptavidin linked to horseradishperoxidase when the probe is biotinylated. Control components mayinclude: normal serum from the animal in which a secondary antibody wasmade; a solution containing nucleolin polypeptide or nucleolin bindingoligonucleotide; a dot blot of nucleolin protein to assaynucleolin-binding reagent reactivity; or fixed or preserved cells thatexpress nucleolin in the plasma membrane. Other components may includebuffers, fixatives, blocking solutions, microscope slides and/or coverslips or other suitable substrates for analysis, such as microtiterplates; detergent or detergent solutions or other cell extractionreagents; miscellaneous reagents, protease inhibitors, variouscontainers and miscellaneous tools and equipment to facilitate theassays.

(a) Containers or Vessels

The reagents included in the kits can be supplied in containers of anysort such that the life of the different components are preserved andare not adsorbed or altered by the materials of the container. Forexample, sealed glass ampules may contain lyophilized nucleolin bindingreagents (such as anti-nucleolin antibodies or nucleolin-bindingoligonucleotides) or buffers that have been packaged under a neutral,non-reacting gas, such as nitrogen. Ampules may consist of any suitablematerial, such as glass, organic polymers (i.e., polycarbonate,polystyrene, etc.), ceramic, metal or any other material typicallyemployed to hold reagents. Other examples of suitable containers includesimple bottles that may be fabricated from similar substances asampules, and envelopes that may have foil-lined interiors, such asaluminum or alloy. Other containers include test tubes, vials, flasks,bottles, syringes, or the like. Containers may have a sterile accessport, such as a bottle having a stopper that can be pierced by ahypodermic injection needle. Other containers may have two compartmentsthat are separated by a readily removable membrane that upon removalpermits the components to mix. Removable membranes may be glass,plastic, rubber, etc.

(b) Instructional Materials

Kits may also be supplied with instructional materials. Instructions maybe printed on paper or other substrate and/or may be supplied as anelectronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, DVD,videotape, audio tape, etc. Detailed instructions may not be physicallyassociated with the kit; instead, a user may be directed to an internetweb site specified by the manufacturer or distributor of the kit, orsupplied as electronic mail.

Methods of Treatment

Therapeutic Methods

Another aspect of the invention pertains to methods of modulatingnucleolin expression or activity for therapeutic purposes. Themodulatory method of the invention involves contacting a cell with anagent that modulates one or more of the activities of nucleolin activityassociated with the cell. An agent that modulates nucleolin activity canbe a nucleic acid or a protein, a naturally occurring cognate ligand ofnucleolin, a peptide, a nucleolin peptidomimetic, or other smallmolecule. Modulatory methods can be performed in vivo (e.g., byadministering the agent to a subject). As such, the invention providesmethods of treating an individual afflicted with a disease or disordercharacterized by aberrant expression or activity of a nucleolin.

Anti-nucleolin Antibodies as Therapeutic Agents

Any antibody, as described in “Detecting nucleolin: antibody-basedmethods: Antibodies,” above, that binds and interferes with nucleolinmay be used to treat tumors and cancers. In certain instances,monoclonal antibodies are preferred as they bind single, specific anddefined epitopes. In other instances, however, polyclonal antibodiescapable of interacting with more than one epitope on nucleolin arepreferred. The antibodies may be whole antibodies and fragments orderivatives thereof. For example, when assaying live cells, using F_(ab)fragments will eliminate cross-linking, thus preventing cells fromendocytosing bound antibodies.

Spliceosome-mediated RNA Trans-splicing (SMaRT) (Mitchell, 1997)

In another embodiment, a subset of cells expressing select members ofnucleolin is targeted through spliceosome-mediated RNA trans-splicing.This method is a means for expressing a heterologous gene in a selectedsubset of cells by targeting a trans-splicing reaction between aprecursor therapeutic molecule (PMT) and pre-mRNA molecules which areuniquely expressed in the specific target cells (Puttaraju, DiPasqualeet al. 2001). The heterologous gene can either be of therapeutic valueto the cell or a toxin which kills the specific cells.

Anti-sense Compounds

Methods of treating a tumor in a subject include administering atherapeutically effective amount of an anti-sense nucleic acid moleculeor ribozymes that may be used to modulate, particularly inhibit, theexpression of nucleolin.

Anti-sense nucleic acid molecules are sequence-specific tools capable ofselectively modifying or silencing gene expression. Anti-sense oligosfunction by binding complementary sequences of a specific gene's cognateRNA by Watson-Crick base pairing to form RNA-oligo hybrid molecules(Knorre and Vlassov 1990). Formation of RNA-oligo hybrids interfereswith RNA function, stability and consequently protein expression.Various mechanisms have been attributed to the inhibition of proteintranslation by anti-sense nucleic acid molecules including: interferenceby physical steric effects and initiation of RNase H-mediateddegradation of the double stranded anti-sense-oligo-probe:mRNA hybrid(Dagle and Weeks 2000). Anti-sense oligonucleotide molecules thus areuseful therapeutically and as a tool to validate drug targets.

A preferred embodiment of a nucleolin anti-sense DNA has at least 10nucleotides, preferably between 15 to 25 nucleotides, or a length thatbinds complementary strands and are most easily formulated and deliveredto target organs and cells. Synthetic anti-sense nucleotides preferablycontain phosphoester analogs, such as phosphorothioate or thioestersrather than entirely natural phosphodiester bonds as these naturallyoccurring bonds are labile to nucleases (Shaw, Kent et al. 1991). Thephosphorothioate class of oligonucleotides have the additionaladvantages of high solubility, ease of synthesis, maintenance ofWatson-Crick nucleotide hydrogen bonding patterns and the ability toactivate RNase H-mediated degradation of cellular mRNA (Stein, Tonkinsonet al. 1991; Crooke 1993; Srinivasan and Iversen 1995; Bock, Griffin etal. 1992).

Ribozymes are enzymatic “catalytic” RNA molecules that are self-cleavingand self-splicing (Cech 1986; Altman 1990; Symons 1992). By combiningcatalytic domains of naturally occurring ribozymes with oligonucleotidesspecific for a target RNA molecule, artificial catalytic RNA moleculesthat cleave specific RNA targets can be made. A ribozyme contains atleast two functional domains: (1) a specialized sequence for RNAspecific binding; and, (2) a catalytic sequence responsible for RNAcleavage (Cech et al., 1992).

Interfering RNA

Tumors and cancers may also be treated by interfering with expression ofkey regulatory genes by administering interfering RNA compositions.Several embodiments of this technology have now been described, such assynthetic interfering RNA duplexes, synthetic short hairpin RNAduplexes, and gene expression systems enabling the in vivo productionand delivery of the interfering RNA molecule (Sharp and Zamore 2000;Bernstein, Caudy et al. 2001; Ketting, Fischer et al. 2001; Sharp 2001;McManus, Petersen et al. 2002; McManus and Sharp 2002; Paddison, Caudyet al. 2002; Paddison, Caudy et al. 2002) (Beach et al., 2001; Fire etal., 2003; Tuschl et al., 2002; Tuschl et al., 2001).

Combination Therapies

In practicing the above-described methods of the present invention, thespecific inhibitors (e.g., antibodies, anti-sense, ribozymes, PMTs orinterfering RNAs, directed against nucleolin) can be used alone or,preferably, in combination with one another, or with other anti-tumoragents such as radiation, chemotherapeutics, and cytotoxic drugs. Suchcombination therapy achieves superior and synergistic therapeuticresults.

Administration

Pharmaceutical Compositions

A “pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration (Remington 2000). Preferred examples ofsuch carriers or diluents include water, saline, Ringer's solutions anddextrose solution. Supplementary active compounds can also beincorporated into the compositions.

General Considerations

A pharmaceutical composition is formulated to be compatible with itsintended route of administration, including intravenous, intradermal,subcutaneous, oral, inhalation, transdermal, transmucosal, and rectaladministration. Solutions and suspensions used for parenteral,intradermal or subcutaneous application can include a sterile diluent,such as water for injection, saline solution, polyethylene glycols,glycerine, propylene glycol or other synthetic solvents; antibacterialagents such as benzyl alcohol or methyl parabens; antioxidants such asascorbic acid or sodium bisulfite; buffers such as acetates, citrates orphosphates, and agents for the adjustment of tonicity such as sodiumchloride or dextrose. The pH can be adjusted with acids or bases, suchas hydrochloric acid or sodium hydroxide. The parenteral preparation canbe enclosed in ampules, disposable syringes or multiple dose vials madeof glass or plastic.

Injectable Formulations

Pharmaceutical compositions suitable for injection include sterileaqueous solutions or dispersions for the extemporaneous preparation ofsterile injectable solutions or dispersion. For intravenousadministration, suitable carriers include physiological saline,bacteriostatic water, CREMOPHOR EL® (BASF; Parsippany, N.J.) orphosphate buffered saline (PBS). In all cases, the composition must besterile and should be fluid so as to be administered using a syringe.Such compositions should be stable during manufacture and storage andmust be preserved against contamination from microorganisms such asbacteria and fungi. The carrier can be a dispersion medium containing,for example, water, polyol (such as glycerol, propylene glycol, andliquid polyethylene glycol), and other compatible, suitable mixtures.Various antibacterial and anti-fungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, and thimerosal, can containmicroorganism contamination. Isotonic agents such as sugars,polyalcohols, such as manitol, sorbitol, and sodium chloride can beincluded in the composition. Compositions that can delay absorptioninclude agents such as aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating theanti-nucleolin agents, and other therapeutic components, in the requiredamount in an appropriate solvent with one or a combination ofingredients as required, followed by sterilization. Methods ofpreparation of sterile solids for the preparation of sterile injectablesolutions include vacuum drying and freeze-drying to yield a solid.

Oral Compositions

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included. Tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,PRIMOGEL®, or corn starch; a lubricant such as magnesium stearate orSTEROTES®; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

Compositions for Inhalation

For administration by inhalation, the compounds are delivered as anaerosol spray from a nebulizer or a pressurized container that containsa suitable propellant, e.g., a gas such as carbon dioxide.

Carriers

In one embodiment, the active compounds are prepared with carriers thatprotect the compound against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable or biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Such materials can beobtained commercially from ALZA Corporation (Mountain View, Calif.) andNOVA Pharmaceuticals, Inc. (Lake Elsinore, Calif.), or prepared by oneof skill in the art.

Transmucosal or Transdermal Formulations

Administration can be transmucosal or transdermal. For transmucosal ortransdermal administration, penetrants that can permeate the targetbarrier(s) are selected. Transmucosal penetrants include, detergents,bile salts, and fusidic acid derivatives. Nasal sprays or suppositoriescan be used for transmucosal administration. For transdermaladministration, the active compounds are formulated into ointments,salves, gels, or creams. Suppositories (e.g., with bases such as cocoabutter and other glycerides) or retention enemas for rectal delivery mayalso be prepared.

Unit Dosage

Oral formulations or parenteral compositions in unit dosage form can becreated to facilitate administration and dosage uniformity. Unit dosageform refers to physically discrete units suited as single dosages forthe subject to be treated, containing a therapeutically effectivequantity of active compound in association with the requiredpharmaceutical carrier. The specification for the unit dosage forms ofthe invention are dictated by, and directly dependent on, the uniquecharacteristics of the active compound and the particular desiredtherapeutic effect, and the inherent limitations of compounding theactive compound.

Dosage

The pharmaceutical composition may further comprise othertherapeutically active compounds as noted herein which are usuallyapplied in the treatment of cancers and tumors.

In the treatment or prevention of conditions which require nucleolinmodulation, an appropriate dosage level of the therapeutic agent willgenerally be about 0.01 to 500 mg per kg patient body weight per daywhich can be administered in single or multiple doses. Preferably, thedosage level will be about 0.1 to about 250 mg/kg per day; morepreferably about 0.5 to about 100 mg/kg per day. A suitable dosage levelmay be about 0.01 to 250 mg/kg per day, about 0.05 to 100 mg/kg per day,or about 0.1 to 50 mg/kg per day. Within this range the dosage may be0.05 to 0.5, 0.5 to 5 or 5 to 50 mg/kg per day. For oral administration,the compositions are preferably provided in the form of tabletscontaining 1.0 to 1000 milligrams of the active ingredient, particularly1.0, 5.0, 10.0, 15.0. 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0,250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0milligrams of the active ingredient for the symptomatic adjustment ofthe dosage to the patient to be treated. The compounds may beadministered on a regimen of 1 to 4 times per day, preferably once ortwice per day.

However, the specific dose level and frequency of dosage for anyparticular patient may be varied and will depend upon a variety offactors including the activity of the specific compound employed, themetabolic stability and length of action of that compound, the age, bodyweight, general health, sex, diet, mode and time of administration, rateof excretion, drug combination, the severity of the particularcondition, and the host undergoing therapy.

Determination of the Biological Effect of the Therapeutic

Suitable in vitro or in vivo assays can be performed to determine theeffect of a specific therapeutic and whether its administration isindicated for treatment of the affected tissue.

In various specific embodiments, in vitro assays may be performed withrepresentative cells of the type(s) involved in the patient's disorder,to determine if a given therapeutic exerts the desired effect upon thecell type(s). Modalities for use in therapy may be tested in suitableanimal model systems including, but not limited to rats, mice, chicken,cows, monkeys, rabbits, dogs and the like, prior to testing in humansubjects. Similarly, for in vivo testing, any of the animal model systemknown in the art may be used prior to administration to human subjects.

The following examples are intended to illustrate the present inventionwithout limitation.

EXAMPLES Example 1 Immunofluorescent Labeling of Plasma MembraneNucleolin in Cells

This example illustrates a procedure that stains nuclear nucleolin, oronly plasma membrane nucleolin.

Cells from the cell lines DU145 (human prostrate cancer), MDA-MB-231(human breast cancer) HeLa (human cervical cancer) and HS27 (normal skinfibroblasts) (all available from ATCC; Manassas, Va.) were released fromculture substrates with trypsin, resuspended into single cells andplated onto microscope slides. The slides seeded with cells wereincubated at 37° C. until they were well attached, as assayed by visualinspection using a microscope. After rinsing the attached cells oncewith PBS for two minutes, they were fixed in 4% formaldehyde/PBS for atleast 15 minutes at 22° C. For nuclear nucleolin staining, cells arepermeabilized with 1% Triton X-100 prior to contacting with antibody.After washing twice with PBS, 5 minutes/wash, non-specific binding siteswere blocked for 15–60 minutes with 1% NGS/PBS at 22° C., and thenincubated with mouse anti-nucleolin antibodies diluted in 1% NGS/PBS orPBS/Tween (0.05%–0.1%) for 1 hour to overnight at 4° C. The samples werewashed four times, 5 minutes each with PBS, and then incubated with goatanti-mouse pAb labeled with FITC-labeled secondary antibodies diluted inPBS for 1 hour at 22° C. After again washing four times with PBS for 5minutes each, the samples were mounted in Mowiol mounting media(prepared as follows: 9 ml/glycerol and 3.36 g Mowiol 40-88 wereagitated for 1 h at 22° C. Then, 9 ml of water was then added, andagitation continued for 2 h at 22° C. Tris (0.2 M, pH 8.5; 18 ml) wasthen added, and the solution incubated for 6 h at 50° C. until thesolids were almost completely dissolved. After centrifugation at 5,000×g, the liquid phase was used for mounting), observed under a microscope,and photographed.

FIGS. 1 and 2 show nuclear (FIG. 1) and plasma membrane (FIG. 2)nucleolin staining in the various cell lines. Shown areimmunofluorescent (FIGS. 1 and 2; panels B, D, F, H) and parallel phasecontrast micrographs (FIGS. 1 and 2; panels A, C, E, G); DU145 cells areshown in A and B; MDA-MB-231 cells are shown in C and D; HeLa cells areshown in E and F; and HS27 cells are shown in G and H. All cell linesshow clear nuclear nucleolin staining (FIGS. 1A, 1C, 1E and 1G). Notethat when the cells are not permeabilized, thus restricting antibodyaccess to the surface plasma membrane, the normal skin cell line, HS27,is completely negative for plasma membrane staining (FIG. 2H) whilecancer cells show strong staining (FIGS. 2B, 2D, 2F and 2H). Stainingplasma membrane nucleolin is thus a superior method for diagnosis andprognosis compared to nuclear nucleolin or silver-staining NORs.

Example 2 Correlation of the Degree of Plasma Membrane NucleolinExpression and Cancer Aggressiveness

This experiment demonstrates that cell lines with high levels of plasmamembrane nucleolin correspond to those with the most rapidproliferation.

Two cancer cell lines, DU145 and HeLa, and one normal cell line, HS27,were assayed for proliferation rate and compared. Cell doubling time iscalculated by determining cell density at regular intervals using theMTT assay (based upon the ability of living cells to reduce 3-(van deLoosdrecht et al., 1994)-2,5 diphenyltetrazolium bromide (MTT) intoformazan; (van de Loosdrecht et al., 1994)), and confirmed by countingthe cells using trypan blue exclusion.

FIG. 3 shows the comparative proliferation rates of DU145 (squares),HeLa (diamonds) and HS27 (circles) as measured by MTT assay. Until 3days of culture, growth rates are similar, but after 3 days, HeLa andDU145 increase at a faster rate than the normal HS27 cells. AlthoughMDA-MB-231 was not included in this experiment, proliferation rate hasbeen determined to be DUI45>MDA-MB-231>HeLa>HS27. Note that the celllines with high levels of plasma membrane nucleolin (see FIG. 2)correspond to those with the most rapid proliferation (DU145 and HeLa).

Example 3 Immunofluorescent Labeling of Nucleolin in Paraffin-embeddedTissue Sections

This example provides a suitable technique to detect and localizenucleolin in a fixed sample that has been embedded.

Sections of cells fixed and embedded in paraffin wax and anchored onmicroscope slides were washed in three changes of xylene (2 minuteseach) to remove the paraffin, hydrated in graded alcohols (series 100%,95% and 70%; 2 minutes each), and placed in PBS for 5 minutes. Antigenrecovery used the approach of low temperature antigen retrieval (LTAR;(Shi et al., 1997; Shi et al., 2001)): After digestion with 0.1%trypsin-EDTA (v/v) (Invitrogen Corp.; Carlsbad, Calif.) diluted in PBSfor 15 minutes at 37° C./5% CO₂, the samples were washed with deionizedwater and incubated in 10 mM citrate buffer (pH 6) for 2 hours at 80° C.After cooling, the slides were rinsed with deionized water and then PBS.

Non-specific binding sites were blocked by incubation in 3% BSA in PBSfor 30 minutes at 22° C. The samples were then incubated with 4 μg/mlmouse anti-nucleolin mAb (Santa Cruz) diluted in PBS/1% NGS at 4° C.overnight. The samples were then brought to 22° C., washed four timeswith PBS for 5 minutes, and then reacted with 50 μg/mlAlexa488-conjugated goat anti-mouse IgG (Molecular Probes; Eugene,Oreg.) and 2 μg/ml propidium iodide diluted in PBS/1% NGS for 1 hour at22° C. After washing four times with PBS for 5 minutes, the samples weremounted in Mowiol mounting medium and observed under a fluorescentmicroscope.

FIG. 4 shows the results of such an experiment. A clinical sample of asquamous cell carcinoma of the head and neck was prepared and probed forplasma membrane nucleolin. Plasma membrane nucleolin signal wasrelegated to malignant, neoplastic cells. FIG. 4A shows theimmunofluorescent signal obtained from probing for nucleolin; the nucleiare counterstained with a DNA-intercalating dye. FIG. 4B shows aparallel phase contrast micrograph. FIGS. 4C and 4D are duplicates ofFIGS. 4A and 4B, except markings have been added to better indicateareas of staining. In region 1, the signal is strong on the cells (faintsignal in relation to the nuclear staining in FIG. 4A); these cells arein loosely-organized tissue and are less densely-packed, suggesting thatthey are malignant. In region 2, normal cells (as delineated bywell-packed cells and organized tissue), cells display no plasmamembrane nucleolin signal.

Example 4 Plasma Membrane Nucleolin Expression in Lung Carcinoma Cells

This example demonstrates that lung carcinoma cells can be easilyidentified by staining for plasma membrane nucleolin.

NCI-H1299 (non-small cell lung carcinoma isolated from H. sapiens lymphnode; (Giaccone et al., 1992; Lin and Chang, 1996)) and NCI-H82 (smallcell lung carcinoma cells, H. sapiens, (Carney et al., 1985; Little etal., 1983; Takahashi et al., 1989)) cells were released from culturesubstrates with trypsin, resuspended into single cells and plated ontomicroscope slides. The cells were incubated at 37° C. until they werewell-attached as assayed by visual inspection using a microscope. Afterrinsing the cells once with PBS for 2 minutes, they were fixed in 4%formaldehyde/PBS for at least 15 minutes at 22° C. After washing twicewith PBS, 5 minutes/wash, non-specific binding sites were blocked for15–60 minutes with 1% NGS/PBS at 22° C., and then incubated with mouseanti-nucleolin antibodies for 1 hour to overnight at 4° C. The sampleswere washed four times, 5 minutes each with PBS and then incubated withgoat anti-mouse pAb labeled with FITC-labeled secondary antibodiesdiluted in PBS with propidium iodide (to stain nuclei) for 1 hour at 22°C. After again washing four times with PBS for 5 minutes each, thesamples were mounted in Mowiol mounting media, observed under amicroscope and photographed.

FIG. 5 shows whole cells probed for plasma membrane nucleolin of the twolung cancer cell lines, NCI-H82 (FIG. 5A; a parallel phase contrastimage is shown in 5B) and NCI-H1299 (FIG. 5C; a parallel phase contrastimage is shown in 5D). In both cell lines, plasma membrane nucleolinstaining is strong; examples of well-stained cells are denoted byasterisk (*) in FIGS. 5A and 5C.

Example 5 Plasma Membrane Nucleolin Staining of Clinical Specimens

To test the feasibility of using this novel method of assaying plasmamembrane nucleolin to diagnose tumor, pre-malignant and malignant cells,clinical specimens from healthy subjects and those suffering from acancer were collected. Samples from peripheral blood, bone marrow andtumor biopsy samples were obtained and stained for plasma membranenucleolin as described in Example 4. FIG. 6 shows phase contrast (B, D,F) and immunofluorescent images (A, C, E) of peripheral blood (A, B) orbone marrow (C, D and E, F). Highly stained cells for plasma membranenucleolin are marked with an asterisk (*); these were only seen in thosepatients suffering from carcinomas (A,B and C,D), while cells from ahealthy patient did not display any plasma membrane staining (E, F).

Example 6 Inhibition of Tumor Growth

A SCID mouse colony was developed using original SCID mice(C.B-17/IcrACSCID) obtained from Taconic (Germantown, N.Y.). The micewere housed in microisolator cages (Allentown Caging Equipment Company,Allentown, N.J.) and maintained under specific pathogen-free conditions.The mice ate NIH31 irradiated pellets (Tekland Premier; Madison, Wis.)and drank autoclaved water. Mice were screened monthly by ELISA serologyfor mycoplasma, mouse hepatitis virus, pinworms, and Sendai virus. Theytested negative.

Female mice 6–8 weeks of age were bled (200 μl) by retro-orbitalpuncture in order to screen for the presence of mouse immunoglobulin(Ig) using ELISA. Only mice with IgG levels <20 μg/ml were used for theexperiments. Mice were weighed once weekly. Tumor cell injections weregiven SC on the mouse's lower right flank in a total volume of 200 μl.Drug injections were administered by intraperitoneal (IP) injection (200μl) when tumors were established (Day 6). As tumors developed, SC tumorswere measured for tumor volume estimation (mm³) in accordance with theformula (a2×b/2) where a is the smallest diameter and b is the largestdiameter. The mice were sacrificed by CO₂ and tumors were harvested.Harvested tumors were sliced into 3 mm sections, set in 10% neutralbuffered formalin for 24 hours, then placed in 70% ethanol, and embeddedin paraffin blocks.

The MDA-MB-231 breast cell line was grown in HyQ RPMI-1640 (1×) media(HyClone, Logan, Utah) with 2.05 mM L-glutamine supplemented with 10%fetal bovine serum (Sigma; St. Louis, Mo.), and maintained in 5% CO2-95%air humidified atmosphere at 37° C. One flask of sub-confluent cells washarvested using 0.25% trypsin-EDTA (HyClone; Logan, Utah) and werecounted using the trypan blue assay technique. The other flasks ofsub-confluent cells were scraped. Cells (95–100% viability) werere-suspended respectively at a concentration of 8×10⁶ cells/200 μl ofsterile saline.

Taxol 10 mg/kg was prepared and administered IP in the volume of 200 μlevery other day for a total of 5 injections in less than an hour fromthe preparation time. The monoclonal and polyclonal antibodies, mouseIgG, and poly rabbit IgG were prepared and injected within an hour IP inthe volume of 200 μl for the initial loading dose of 10 mg/kg. Theremaining amount of antibodies were prepared at a maintenance dose of 3mg/kg in 200 μl, which was aliquoted and frozen into 6 separate tubesper antibody for weekly injections for a total of 6 weeks. Each weeklymaintenance dose was thawed and injected within an hour. The PBS 1 ×control was administered IP in 200 μl weekly for a total of 7injections. Results of the experiment demonstrate significantly greatertumor regression with the combination of any antibody composition andTaxol.

Example 7 (Prophetic) Correlation of the Degree of Plasma MembraneNucleolin Expression and Cancer Aggressiveness

Thirty-three lung carcinoma cell lines are analyzed, mostly availablefrom the American Type Culture Collection (Manassas, Va.). Cell doublingtime is calculated by determining cell density at regular intervalsusing the MTT assay and confirmed by counting the cells using trypanblue exclusion. In each experiment HeLa cells (Gey et al., 1952) areincluded as an internal control. Each value is determined from at leasttwo independent experiments with triplicate samples. To determine levelsof nuclear and plasma membrane nucleolin, two methods are implemented.First, nuclear and plasma membrane extracts are prepared from each cellline using methods that have as described (Ausubel et al., 1987; Bateset al., 1999; Yao et al., 1996a). Briefly, cells are harvested andresuspended in a hypotonic buffer, then allowed to swell on ice forseveral minutes. Cells are lysed using a Dounce homogenizer, and nucleiare collected by centrifugation. Nuclei are resuspended in a high saltbuffer to extract nuclear proteins; salt is then removed by dialysis.Plasma membrane proteins can be isolated from the S-100 fraction and areseparated from cytosolic proteins and other organelles by centrifugationthrough a sucrose gradient. Nuclear and PM extracts from different cellare analyzed by Western blot analysis (Ausubel et al., 1987) using ananti-nucleolin antidody (Santa Cruz) followed by chemiluminescentvisualization. Nucleolin levels are then quantified by densitometry ofthe resulting signal recorded on X-ray film and normalized to theintensity of HeLa extract controls. The second approach to determinenucleolin levels involves immunofluorescent probing of the cell linesfor nucleolin. Cells are probed for nucleolin surface expression inparallel with DU145 cells (Mickey et al., 1977; Stone et al., 1978) as apositive control, HS27 cells as a negative control and HeLa cells as areference (see FIG. 2). Cells are photographed and ranked in order ofdegree of signal, which may also be quantified (using systems that usesoftware and images to quantitate pixels; in this instance, video imagesare used)or qualitatively evaluated. The data are then subjected tostatistical analysis to demonstrate correlations with the degree of cellproliferation (higher rates of cell proliferation indicate moreaggressive cancer cells) with the intensity of nucleolin signal acrossthe entire sample and within subsets.

Example 8 (Prophetic) Lung Cancer Detection

In this example, patient biopsies, sputum samples and resected lungtissue are probed for plasma membrane nucleolin, and these results arecompared to other diagnostic and prognostic markers for lung cancer,utilizing archival and routine clinical specimens for this study.

Methods

Specimens including bronchial biopsies, sputum samples, and resectedlung tissue are obtained from human subjects, both healthy and thosesuffering from lung cancer, and each sample encoded such that at thetime of nucleolin probing and observation, the sample origin is unknown.

Probing these samples using immunohistochemical techniques are thenimplemented. For example, plasma membrane nucleolin is probed with oneor more anti-nucleolin Abs selected from Table 1, a signal generatedfrom a flourophore-tagged secondary Ab, and the samples observed andphotographed. Appropriate controls include probing with the secondaryantibody only, probing with no antibodies, probing with pre-immune serumonly, and probing with an antibody known not to react with the celltypes being analyzed. To facilitate visualization and localizationdetermination, the cells can be counterstained with Hoechst 33258 orpropidium iodide (to visualize nuclei) and/or with fluorescent-taggedphalloidin or phallicidin (to visualize the actin cytoskeleton). Thesamples are observed, scored (surface signal indicating plasma membranenucleolin expression) and documented.

All cited publications are incorporated herein by reference. The termsand expressions which have been employed in the present disclosure areused as terms of description and not of limitation, and there is nointention in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof;various modifications are possible and are within the scope of theinvention.

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1. A method of treating cancer in a mammal comprising administering tosaid mammal a therapeutically effective amount of an anti-nucleolinagent and a pharmaceutically acceptable carrier; wherein theanti-nucleolin agent comprises a nucleolin monoclonal antibody.
 2. Themethod of claim 1, further comprising administering to said mammal achemotoxic or chemotherapeutic agent.
 3. The method of claim 1, furthercomprising treating said mammal with radiation therapy.
 4. The method ofclaim 2, wherein said chemotoxic or chemotherapeutic agent is selectedfrom the group consisting of cyclophosphamide, etoposide, doxorubicin,methotrexate, vincristine, procabazine, prednizone, dexamethasone,tamoxifen citrate, carboplatin, cisplatin, oxaliplatin, 5-fluorouracil,camptothecin, zoledronic acid, Ibandronate and mytomycin.
 5. A method oftreating cancer in a human, comprising administering to said human atherapeutically effective amount of an anti-nucleolin monoclonalantibody and a pharmaceutically acceptable carrier, wherein saidnucleolin antibody is substantially non-immunogenic to human.
 6. Themethod of claim 5, further comprising administering to said human achemotoxic or chemotherapeutic agent.
 7. The method of claim 5, furthercomprising treating said human with radiation therapy.
 8. The method ofclaim 6 wherein said chemotoxic or chemotherapeutic agent is selectedfrom the group consisting of cyclophosphamide, etoposide, doxorubicin,methotrexate, vincristine, procabazine, prednizone, dexamethasone,tamoxifen citrate, carboplatin, cisplatin, oxaliplatin, 5-fluorouracil,camptothecin, zoledronic acid, Ibandronate and mytomycin.
 9. A method oftreating cancer in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin monoclonalantibody, a chemotoxic or chemotherapeutic agent and a pharmaceuticallyacceptable carrier.
 10. The method of claim 9, wherein said chemotoxicor chemotherapeutic agent is selected from the group consisting ofcyclophosphamide, etoposide, doxorubicin, methotrexate, vincristine,procabazine, prednizone, dexamethasone, tamoxifen citrate, carboplatin,cisplatin, oxaliplatin, 5-fluorouracil, camptothecin, zoledronic acid,Ibandronate and mytomycin.
 11. A method of treating cancer in a mammalcomprising administering to said mammal a therapeutically effectiveamount of an anti-nucleolin monoclonal antibody and a pharmaceuticallyacceptable carrier, and further treating said mammal with radiationtherapy.
 12. The method of claim 2, further comprising treating saidmammal with radiation therapy.
 13. The method of claim 4, furthercomprising treating said mammal with radiation therapy.
 14. The methodof claim 6, further comprising treating said human with radiationtherapy.
 15. The method of claim 8, further comprising treating saidhuman with radiation therapy.
 16. The method of claim 9, furthercomprising treating said mammal with radiation therapy.
 17. The methodof claim 10, further comprising treating said mammal with radiationtherapy.
 18. The method of claim 1, wherein the therapeuticallyeffective amount of an anti-nucleolin agent and a pharmaceuticallyacceptable carrier comprises an injectable formulation.
 19. The methodof claim 5, wherein the therapeutically effective amount of ananti-nucleolin monoclonal antibody and a pharmaceutically acceptablecarrier comprises an injectable formulation.
 20. The method of claim 9,wherein the therapeutically effective amount of an anti-nucleolinmonoclonal antibody, a chemotoxic or chemotherapeutic agent and apharmaceutically acceptable carrier comprises an injectable formulation.21. The method of claim 11, wherein the therapeutically effective amountof an anti-nucleolin monoclonal antibody and a pharmaceuticallyacceptable carrier comprises an injectable formulation.